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A feeding strategy that maintains high content of functional molecules in buffalo milk has been verified by giving Sorghum vulgare as green fodder, but it is not available all year round. The aim of this study was to evaluate the inclusion of former food products (FFPs) containing 87% biscuit meal (nonstructural carbohydrate: 60.1%; starch 14.7; crude protein 10.6), in the diet of buffaloes in terms of: (a) fermentation characteristics through gas production technique; (b) milk yield (MY) and quality; (c) content of some biomolecules and total antioxidant activity. The experiment was performed involving 50 buffaloes divided into two groups: Green group and FFPs group (animals fed Total Mixed Ration with either green forage or FFPs respectively). Daily MY was recorded and milk qualitative analyses were determined monthly for 90 days. Furthermore, fermentation characteristics of the diets were studied in vitro. No significant differences were recorded in feed intake, BCS and MY and quality. Similar in vitro fermentation data of two diets were found, with slight differences in terms of gas production and degradability. During the incubation, kinetic parameters showed a faster fermentation process with the diet of the FFPs group in relation to Green group (p < 0.05). Green group had higher levels (p < 0.01) of γ‐butyrobetaine, glycine betaine, l‐carnitine and propionyl l‐carnitine in milk, whereas no differences were observed for δ‐valerobetaine and acetyl l‐carnitine. Total antioxidant capacity and iron reduction antioxidant assay were higher (p < 0.05) in the plasma and milk of the Green group. The administration of a diet high in simple sugars, obtained with FFPs, seems to favour the ruminal biosynthesis of some metabolites in milk, such as δ‐valerobetaine and acetyl‐ l‐carnitine, similar to green forage administration. Overall, the use of biscuit meal can be an alternative to green fodder when it is not available to ensure environmental sustainability and optimize costs without compromising milk quality. [ABSTRACT FROM AUTHOR]

The study evaluated the effects of eCG treatment prior to ovum pick up (OPU) on follicular population, oocyte and embryo yields in summer and autumn in Podolic cattle. The effects of repeated OPU on cattle wellbeing was also documented. Twenty-six animals were used, and split into two groups, treatment (OPU; n = 18) and control (CG; n = 8). The OPU cattle were subsequently split into two subgroups (n = 9) and underwent repeated OPU, without and with eCG priming, for a total of 8 sessions (4 sessions/season). Follicular population, oocyte and embryo yields were recorded in those sub-groups. CG was handled in the same manner of OPU, except for epidural anaesthesia and follicular aspiration. Biochemical profile, serum protein electrophoresis and haptoglobin levels were analysed in OPU and CG. Hormonal priming increased the number of medium follicles (1.7 ± 0.2 vs 0.9 ± 0.2, p <.05), while it decreased the recovery rate and number of cumulus-enclosed oocytes (COCs) (recovery rate: 38.3 ± 3.5 vs 60.5 ± 4.0%; COCs: 2.3 ± 0.3 vs 3.4 ± 0.4, respectively; p <.01). However, priming increased cleavage (72.9 ± 5.7 vs 49.4 ± 5.4; p <.05) and blastocyst (41.1 ± 5.7 vs 23.0 ± 4.2; p =.054) rates. With regard to season's effect, a higher number of COCs was recorded in autumn than in summer (3.1 ± 0.4 vs 2.6 ± 0.3; p <.05) without affecting though the number of embryos produced (0.9 on average). Since haematological parameters did not vary between OPU and CG, our preliminary data suggest that repeated OPU may be used as a conservation strategy in Podolic cattle without affecting wellbeing. Podolic cattle is an endangered Italian breed, reared in semi-extensive/extensive systems Ovum pick-up (OPU) can be carried out in this breed without impairing animal health and welfare The eCG treatment before OPU (priming) did not improve the number of embryos produced per donor. [ABSTRACT FROM AUTHOR]

Artificial insemination (AI) is important for genetic improvement and to control the period of breeding in buffalo and has increased significantly over the past 20 years. AI is more difficult in buffalo compared with cattle due to variable estrous cycles, reduced estrous behavior, and reproductive seasonality. The latter is associated with a higher incidence of anestrus and increased embryonic mortality during the nonbreeding season. Protocols to control the stage of the estrous cycle have undergone recent development in buffalo. These protocols are based on the control of both the luteal phase of the cycle, mainly by prostaglandins and progesterone, and follicle development and ovulation, by prostaglandins, progesterone, GnRH, hCG, eCG and estradiol. Protocols that synchronize the time of ovulation enable fixed timed AI, avoiding estrous detection. Factors to consider when selecting an AI protocol include animal category (heifers, primiparous or pluriparous), reproductive status (cyclic or anestrus), and season. This review looks at the current status of estrus synchronization and AI in buffalo and provides some practical suggestions for application of AI in the field. • Artificial Insemination (AI) can guarantee genetic improvement in buffalo. • The reproductive peculiarities of the species impair the use of AI in buffalo. • Satisfying results may be obtained by the use of synchronization treatments. • Animal category, reproductive status and season have to be examined when applying AI. • Re-synchronization treatments improve the reproductive performance of the herd. [ABSTRACT FROM AUTHOR]

The combined effect of six consecutive timed artificial inseminations (TAIs) on pregnancy rates, following two different synchronization protocols on buffalo heifers, over a period of seven months typically characterized by low breeding performances, were investigated in this study. A total of 2189 TAIs were performed on 1463 buffalo heifers within a large buffalo farm in the south of Italy. Individual animals were allowed to undergo synchronization protocol (either a slightly modified Ovsynch or Progesterone treatment) and TAI until establishment of pregnancy or else for not more than six consecutive times. Semen of seven proven bulls was used throughout the study, which was carried out from March to September of the same year. Therefore, other than the effect given by consecutive TAIs over time, a monthly and a seasonal effect could also be tested, once the entire period was split into a Low Breeding Season (LBS) from March to June, and a Transition to Breeding Season (TBS) from July to September. From the data recorded in this study and the statistical analysis performed, it can be stated that the two protocols for the synchronization of ovulation were similar in efficiency in determining pregnancies with an overall fertility rate of 89.4% when the comparison was run both on a monthly basis or when months were grouped into two different seasons. In addition, an average of 1.83 AI/pregnancy was reported, slightly higher for the Ovsynch when compared to the Progesterone protocol: 1.91 vs 1.70, respectively. Finally, when considering the number of progressive synchronization treatments implemented over time as covariate, neither Ovsynch nor Progesterone treatment significantly affected pregnancy rates following the first of the six synchronization sessions. However, repeating the synchronization procedure, the progesterone based protocol resulted in significantly higher probability of success in terms of established pregnancies during the second and third re-synchronization sessions. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the relationship between the ovarian follicular response at the start of the OVsynch–fixed-time artificial insemination (TAI) program and pregnancy outcome in the Mediterranean river buffalo. The study involved 116 pluriparous buffaloes at 118 ± 65 days in milk maintained under commercial conditions on a buffalo dairy enterprise in southern Italy. Synchronization of the stage of the estrous cycle in the Ovsynch–TAI program involves an injection of GnRH on Day 0 (GnRH1), PGF 2α on Day 7, and GnRH on Day 9 (GnRH2). Timed artificial insemination is carried out on Day 10. Ovarian ultrasound examinations were undertaken on days 0, 2, 7, 9, 10, and 11 to record total follicles, the dimensions of follicles greater than 0.75 cm, dimensions of the corpus luteum (CL), and the occurrence of ovulation. Blood concentrations of P4 were measured on days 0 and 10 and 10 days after TAI. Pregnancy status was determined on days 27 and 45 after TAI to calculate late embryonic mortality. The data were analyzed by ANOVA and multiple logistic regression. A total of 70 of 116 (60.4%) buffaloes showed ovulation after GnRH1. Buffaloes that ovulated after GnRH1 had a smaller (P < 0.05) area of the ovulatory follicle on Day 0 compared with buffaloes that did not ovulate (1.08 ± 0.1 and 1.29 ± 0.1 cm 2 , respectively). The former buffaloes also had a larger (P < 0.05) CL area (1.81 ± 0.1 and 1.51 ± 0.1 cm 2 , respectively) and greater (P < 0.05) blood concentrations of P4 (1.84 ± 0.2 and 1.34 ± 0.2 ng/mL, respectively) on Day 0. A larger (P < 0.05) proportion of buffaloes that ovulated after GnRH1 showed regression of the CL after the injection of PGF 2α on Day 7 compared with buffaloes that did not ovulate (68 of 69 [98.6%] and 27 of 32 [84.4%], respectively). Buffaloes that ovulated after GnRH1 also had a greater (P < 0.05) ovulation rate after GnRH2 than buffaloes that did not ovulate (65 of 70 [92.8%] and 37 of 46 [80.4%], respectively). Buffaloes that ovulated after GnRH1 had a higher (P < 0.01) pregnancy rate than buffaloes that did not ovulate, both at Day 27 (53 of 70 [75.7%] and 14 of 46 [30.4%], respectively) and Day 45 (46 of 70 [65.7%] and 10 of 46 [21.7%], respectively). The incidence of late embryonic mortality tended to be less for buffaloes that ovulated after GnRH1 than buffaloes that did not ovulate (7 of 53 [13.2%] and 4 of 14 [28.6%], respectively). The present findings have demonstrated a strong direct relationship between the ovarian follicular response at the start of an OVsynch–TAI program and pregnancy outcome in the Mediterranean river buffalo. [ABSTRACT FROM AUTHOR]

River buffalo are able to adapt to diverse climatic zones and are widespread globally. The resource use efficiency of buffalo is highly relevant in a resource-constrained world and the increasing requirement to produce more food. Buffalo clearly have an important role in meeting the growing demand for animal protein. In the Mediterranean and higher latitudes, buffalo show annual cycles of ovarian activity, embryonic development, and pregnancy rate. In buffalo, the CL starts to develop early in the cycle, and there is also an early increase in concentrations of progesterone (P 4 ) in circulation. This appears to be necessary for optimal embryonic development. The failure to establish a pregnancy in buffalo can occur before Day 21 (early embryonic mortality), from Day 21 to 45 (late embryonic mortality), and from Day 46 to 90 (fetal mortality) after mating. Treatment with P 4 , hCG, and GnRH on Day 5 after mating increases P 4 in circulation and reduces early embryonic mortality in circumstances where concentrations of P 4 are relatively low. The same treatments applied on Day 20 to 25 after mating can lower the occurrence of late embryonic mortality and fetal mortality. [ABSTRACT FROM AUTHOR]

A detailed study on the structure and function of the CL in the Day-5 to Day-10 window of development, and relationship to the likelihood of pregnancy, was undertaken in Italian Mediterranean buffaloes. In experiment 1, buffaloes underwent synchronization of estrus and fixed-time artificial insemination (n = 23). Features of the CL were measured from Days 5 to 10 after fixed-time artificial insemination, and pregnancy was confirmed on Day 70. Buffaloes that established a pregnancy (n = 14) had a larger CL area (1.31 ± 0.1 vs. 1.09 ± 0.1 cm 2 ; P < 0.01) and greater progesterone (P 4 ) concentrations (1.90 ± 0.1 vs. 1.48 ± 0.1 ng/mL; P < 0.01) during Days 5 to 10 compared with nonpregnant buffaloes. In the same period, blood flow measured as time average medium velocity tended to be greater (P = 0.059) in buffaloes that were subsequently pregnant versus nonpregnant buffaloes (10.8 ± 0.8 vs. 8.4 ± 0.9). There was a relationship ( R 2 = 0.136; P < 0.05) between CL area, P 4 , and time average medium velocity from Days 5 to 10. Logistic regression analysis showed that P 4 concentration on Day 10 had a significant influence on pregnancy (odds ratio, 19.337; P < 0.01). In experiment 2, highly vascularized CLs (HVCLs, n = 3) and lowly vascularized CLs (LVCLs, n = 3) on Day 5 were examined by contrast-enhanced ultrasonography and then subjected to histologic investigation. Blood flow was greater in HVCLs than in LVCLs. Highly vascularized CLs showed intense staining for factor VIII and had many small, irregular-shaped blood vessels, whereas LVCLs had low factor VIII staining and relatively few large, regular-shaped vessels. Luteal cell expression of vascular EGF was greater for HVCLs compared with LVCLs. The study has shown that greater development and function of the CL from as early as Day 5 is related to an increased likelihood of pregnancy in the buffalo. Corpus lutea that show early development at Day 5 have greater expression of vascular EGF and factor VIII, increased vascularization, and higher blood flow. [ABSTRACT FROM AUTHOR]

The aim of this study was to quantify nitrogen (N) and phosphorus (P) utilisation and excretion in intensive dairy buffaloes. Italian Mediterranean buffaloes were divided into 4 groups (n=6/group) as follows: Groups M50, M125 and M225 at 50, 125 and 225 days of lactation, respectively, and Group NL which was non-lactating. Lactating buffaloes had greater (P<0.05) dry matter and organic matter intake than NL buffaloes. Buffaloes in Group M50 had a lower (P<0.05) crude protein (CP) apparent digestibility than buffaloes in Groups M125 and M225. Group NL had a higher (P<0.01) real CP digestibility than Group M50. Buffaloes in Groups M50 and NL had a negative N balance (-79 and -12 g, respectively) whilst buffaloes in Groups M125 and M225 had a positive N balance (26 and 16 g, respectively). Buffaloes in Group M50 had more (P<0.05) N in urine (204 g) than Group NL (87 g). Buffaloes in Group NL had less (P<0.01) P in both faeces (12.9 g) and urine (0.8 g) compared with the three groups of lactating buffaloes combined (25 g in faeces and 12 g in urine) and they also had greater (P<0.05) P digestibility (68%) than lactating buffaloes combined (43%). The present study has shown that buffaloes have a relatively high efficiency of N and P utilisation. [ABSTRACT FROM AUTHOR]

Background Brucellosis is considered the world's most widespread zoonotic infection. It causes abortion and sterility in livestock leading to serious economic losses and has even more serious medical impact in humans, since it can be a trigger to more than 500,000 infections per year worldwide. The aim of this study was to evaluate the role of Haematopinus tuberculatus, a louse that can parasitize several ruminants, as a new host of brucellosis. Louse specimens were collected from seropositive and seronegative water buffaloes and divided in 3 developmental stages: adults, nymphs and nits. All samples were separately screened for Brucella spp. DNA and RNA detection by Real Time PCR. In particular, primers and probes potentially targeting the 16S rRNA and the Brucella Cell Surface 31kDalton Protein (bcsp31) genes were used for Real Time PCR and buffalo β actin was used as a housekeeping gene to quantify host DNA in the sample. A known amount of B. abortus purified DNA was utilized for standard curve preparation and the target DNA amount was divided by the housekeeping gene amount to obtain a normalized target value. A further molecular characterization was performed for Brucella strain typing and genotyping by the Bruce-ladder, AMOS-PCR and MLVA assays. Data were statistically analysed by ANOVA. Results Brucella abortus DNA and RNA were detected in all developmental stages of the louse, suggesting the presence of viable bacteria. Data obtained by MLVA characterization support this finding, since the strains present in animals and the relative parasites were not always identical, suggesting bacterial replication. Furthermore, the detection of Brucella DNA and RNA in nits samples demonstrate, for the first time, a trans-ovarial transmission of the bacterium into the louse. Conclusions These findings identified H. tuberculatus as a new host of brucellosis. Further studies are needed to establish the role of this louse in the epidemiology of the disease, such as vector or reservoir. [ABSTRACT FROM AUTHOR]

The sucking louse Haematopinus tuberculatus (Burmeister 1839) is an ectoparasite of buffaloes, cattle, camels, and American bison. Alphacypermethrin (ACYP) is a pyrethroid insecticide commonly used to control arthropods of veterinary and public health interest. Therapeutics, such as antiparasitic compounds, is often administered to buffaloes based on dosage and intervals recommended for cattle because very few drugs have buffalo-specific label indications. A trial was conducted on 20 louse-infested buffaloes at a farm to assess the efficacy and safety of ACYP pour-on, at the manufacturer's recommended dose for cattle, on buffaloes naturally infested by H. tuberculatus. Ten animals were assigned to ACYP-treated group (ACYP-group) and ten to untreated control group (C-group). On day 0, all ACYP-group buffaloes received alphacypermethrin pour-on. Louse counts were performed on days −1, 7, 14, 21, 28, 35, 42, 49, and 56 at eight predilection sites on the skin of each buffalo. ACYP was completely effective (100 %) at day 7, highly effective (99.8 %) at day 14, and completely effective (100 %) from day 21 until the end of the study (day 56 post-treatment). During the trial, ACYP was well tolerated by all animals as there were no observed clinically adverse reactions. The results of this trial suggest that ACYP is an effective, safe, and user-friendly compound suitable for treatment of buffaloes with natural louse infestations. [ABSTRACT FROM AUTHOR]

The chewing louse Werneckiella equi is an ectoparasite of donkeys and other equids. Alphacypermethrin (ACYP) is a pyrethroid insecticide commonly used for the control of insects of veterinary and public health concerns. A trial was conducted to assess the efficacy of ACYP against W. equi on naturally infested donkeys. Parasitological investigations were performed on 13 animals. On day 0, the donkeys received ACYP pour-on at the manufacturer's recommended dose rate for cattle. Louse counts were performed on days −1, 7, 14, 21, 28, 35, 42, 49, and 56 at seven predilection sites on the skin of each donkey. ACYP was completely effective (100 %) from day 7, until the end of the study. ACYP was well tolerated by all animals as there were no observed clinical adverse reactions. ACYP was highly effective, safe, user-friendly, and considered to be suitable for the treatment of donkeys for W. equi infestation. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to evaluate the effect of an Ovum Pick-up (OPU) treatment carried out for 9 months in buffalo (Bubalus bubalis) species. Eight pluriparous non-lactating buffalo cows underwent OPU for 9 months. Recovered cumulus enclosed oocytes (COCs) were classified and COCs suitable for in vitro embryo production (IVEP) were in vitro matured (IVM), fertilized (IVF) and cultured (IVC) to the blastocyst (Bl) stage. Animals were monitored for a total period of 270 days, but at the summer solstice, follicular turnover decreased and at the 68-day of the trial, we decided to increase the OPU sampling interval from 3–4 to 7 days. It was therefore possible to distinguish two phases: a first phase (18 sessions), during which OPU was carried out twice weekly and a second phase (16 sessions) during which OPU sessions were performed weekly. This reduction did not modify the percentage of good quality COCs, while the incidence of grade D COCs decreased (P <0.01). Furthermore, embryo production was higher in the second phase, either if embryos were calculated on the total recovered COCs (8.3% vs. 21.4%; P <0.01) and on grade A+B COCs (13.0% vs. 32.1%; P <0.01), that supposedly should have given similar blastocyst yield. During the total period of the trial it was possible to distinguish a first period of 6 months (34 sessions) characterized by blastocyst production (0.36 blastocyst/buffalo/session), followed by an unproductive period of 3 months (12 sessions), during which embryos were not produced. During the first 6 months a higher (P <0.01) number of follicles (5.06 vs. 3.71), small follicles (3.38 vs. 2.07), total COCs (2.58 vs. 1.56) and good quality (A+B) COCs (1.51 vs. 0.94) per subject/session were recorded compared to the last 3 months. No Blastocyst were produced during the second period, even if the percentage of grade A+B COCs was similar to that recorded during the first period. In conclusion, buffalo cows submitted to repeated OPU sampling for a 9-month period, showed a decline of follicle recruitment and oocyte collection after the first two months of samplings. After 6-month of samplings, in spite of the quality grade of the collected oocytes, we found a drop in their developmental competence. [Copyright &y& Elsevier]

Abstract: The aim was to determine the factors that contribute to embryonic mortality in buffaloes mated by AI during a period of increasing day length which corresponds to a natural decline in reproductive activity. Italian Mediterranean buffalo cows (n =243) showing regular estrous cycles were synchronized using the Ovsynch-TAI program and mated by AI at 16 and 40h after the second injection of GnRH. Blood samples were collected on Days 10 and 20 after the first AI and assayed for progesterone (P 4). Pregnancy diagnosis was undertaken on Days 26 and 40 after the first AI using rectal ultrasonography. Buffaloes with a conceptus on Day 26 but not on Day 40 were judged to have undergone embryonic mortality and for these animals uterine fluid was recovered by flushing and analysed for common infectious agents. Estrus synchronization was achieved in 86% of buffaloes and the pregnancy rate on Day 40 was 34%. Embryonic mortality between Days 26 and 40 occurred in 45% of buffaloes and was associated with the presence of significant infectious agents in only 10 buffaloes (8%). Concentrations of P 4 on Day 10 after AI were higher (P &#60;0.05) in buffaloes that established a pregnancy than in buffaloes that showed embryonic mortality that was not associated with infectious agents. Similarly, on Day 20 after AI P 4 concentrations were higher (P &#60;0.01) in pregnant buffaloes compared with non-pregnant buffaloes and buffaloes that had embryonic mortality. It is concluded that a reduced capacity for P 4 secretion can explain around 50% of embryonic mortalities in buffaloes synchronised and mated by AI during a period of low reproductive activity and that other as yet unidentified factors also have a significant effect on embryonic survival. [Copyright &y& Elsevier]

The aim in this study was to compare two estrus synchronization protocols in buffaloes. Animals were divided into two groups: Group A (n=111) received 100 μg GnRH on Day 0, 375 μg PGF2α on Day 7 and 100 μg GnRH on Day 9 (Ovsynch); Group B (n=117) received an intravaginal drug release device (PRID®) containing 1.55 g progesterone and a capsule with 10 mg estradiol benzoate for 10 days and were treated with a luteolytic dose of PGF2α and 1000 IU PMSG at the time of PRID® withdrawal. Animals were inseminated twice 18 and 42 h after the second injection of GnRH (Group A) and 60 and 84 h after PGF2α and PMSG injections (Group B). Progesterone (P4) concentrations in milk samples collected 12 and 2 days before treatments were used to determine cyclic and non-cyclic buffaloes, and milk P4 concentrations 10 days after Artificial insemination (AI) were used as an index of a functional corpus luteum. Cows were palpated per rectum at 40 and 90 days after AI to determine pregnancies. All previously non-cyclic animals in Group B had elevated P4 (>120 pg/ml milk whey) on Day 10 after AI. Accordingly, a greater (P<0.01) relative percentage of animals with elevated P4 10 days after AI were observed in Group B (93.2%) than in Group A (81.1%). However, there was no difference in overall pregnancy rates between the two estrus synchronization protocols (Group A, 36.0%; Group B 28.2%). When only animals with elevated P4 on Day 10 after AI were considered, pregnancy rate was higher (P<0.05) for animals in Group A (44.4%) than Group B (30.3%). The findings indicated that treatment with PRID® can induce ovulation in non-cyclic buffalo cows. However, synchronization of estrus with Ovsynch resulted in a higher pregnancy rate compared with synchronization with PRID®, particularly in cyclic buffalo. [Copyright &y& Elsevier]

This study was undertaken in order to evaluate the effect of oocyte source (live animals and abattoir ovaries) on subsequent embryo development in buffalo (Bubalus bubalis). Cow ovaries were also collected as oocyte donors for in vitro embryo production (IVEP).Three hundred thirty-eight oocytes were recovered by ovum pick up (OPU, Group A) from 8 pluriparous buffalo cows, while 1127 and 1457 oocytes were aspirated, respectively, from buffalo (Group B) and bovine (Group C) slaughterhouse ovaries. Cumulus enclosed oocytes (COCs) suitable for IVEP were in vitro matured (IVM), fertilized (IVF) and cultured (IVC) to the tight morula (Tm) and blastocyst (Bl) stage.Within buffalo species Group A had a higher Bl yield (29.7 % versus 19.9%; P<0.05) and a lower proportion of embryos arrested at Tm stage (11.1% versus 22.3%; P<0.05) than Group B.Within slaughterhouse groups cattle oocytes had a higher cleavage rate (83.9% versus 64.8%; P<0.05) and yielded 49.2% more blastocysts than buffalo. However, when data are related to the total number of cleaved oocytes, only 13.7% more blastocysts were produced in cattle than in buffalo.In conclusion, in buffalo species the source of oocytes significantly affected post-fertilization embryo development, as demonstrated by the higher Bl yields derived from OPU-derived oocytes.A higher overall IVEP efficiency, mainly related to the higher cleavage rate, was recorded in cattle compared with buffalo when ovaries from an abattoir were used as oocyte donors. [Copyright &y& Elsevier]

• Green feed increases concentration of health-promoting molecules in buffalo milk. • Green feed increases the antioxidant activity of buffalo milk. • Green feed increases the antineoplastic of buffalo milk. • Increased biomolecules support buffalo milk consumption within a balanced diet. The effect of green feed on health-promoting biomolecules in milk was examined in dairy buffaloes. Buffaloes received a total mixed ration (TMR) (Control, C; n = 40) or TMR + alfalfa green feed (30% of diet) (Treated, T; n = 40). Biomolecules and functional activity were measured in milk obtained twice-monthly. Treated buffaloes had higher milk l -carnitine, acetyl- l -carnitine, propionyl- l -carnitine and δ-valerobetaine (P < 0.01). They also had higher antioxidant activity (P < 0.01). Compared with C buffaloes, milk of T buffaloes improved the viability of endothelial cells exposed to high-glucose (P < 0.01), and reduced intracellular lipid peroxidation, reactive oxygen species (ROS), and cytokine release (P < 0.01). Milk of T buffaloes inhibited with greater potency the viability of human HCT116 and Cal 27 cancer cells (P < 0.001). The findings show that including green feed in the diet of dairy buffaloes enhances health-promoting biomolecules and the antioxidant and antineoplastic properties of milk. [ABSTRACT FROM AUTHOR]

Milk somatic cell count (SCC), an indicator of udder health and milk hygiene, has not been implemented either in milk payment systems or in the current selection index of Italian buffalo so far. As a matter of fact, there is room to improve udder health through genetic and management strategies in this species. Repeated milk SCC test-day (TD) records on the same animal are useful to derive novel phenotypes on lactation basis by using somatic cell score (SCS) and thus to disclose buffaloes with udder issues to be monitored. In this study, sources of variation of SCC-derived traits in buffalo milk were investigated and their effect on milk yield and composition traits was estimated. Mean SCS (SCS150), standard deviation of SCS (SCS_SD150) and severity (SEV150, ratio between the number of TD SCC >200,000 cells/mL and total number of TD available in the first 150 DIM) in the first 150 days in milk were calculated using TD data of 45,312 lactations from 35,623 buffaloes. Sources of variation of such traits were investigated through a linear model. Both SCS150 and SEV150 increased with parity, whereas SCS_SD150 decreased (p <.001). Subsequently, the three traits were separately included in the model as explanatory variables to estimate their effect on milk yield and composition traits. Results showed that milk yield and lactose content were lower in animals with high and variable SCS in the first 150 days of lactation (p <.001). This study opens the debate on the development of an udder health index for the Italian buffalo. So far, somatic cell count has not been considered in payment systems of buffalo milk in Italy. There is need to improve mastitis resistance and udder health in dairy buffaloes. Traits derived from test-day records can be used for management and breeding purposes in buffaloes. [ABSTRACT FROM AUTHOR]

Seasonal breeding in buffalo is influenced by exogenous (photoperiod, climate, nutrition, management) and endogenous (hormones, genotype) factors. Buffalo are negatively photoperiodic and show a natural increase in fertility during decreasing day length. The hormone melatonin is produced by the pineal gland and has a fundamental role in photoperiodic time measurement within the brain. This drives annual cycles of gonadotropin secretion and gonadal function in buffaloes. Some melatonin is released into the systemic circulation and, together with peripherally produced melatonin, acts at somatic tissues. In the ovaries and testes of buffalo, melatonin acts as an antioxidant and scavenges oxygen free radicals to reduce both oxidative stress and apoptosis. This has beneficial effects on gametogenesis and steroidogenesis. Female buffalo treated with melatonin show an improved response to estrus synchronization protocols in out-of-season breeding. Melatonin acts through melatonin receptors MT 1 and MT 2 and the gene for MT 1 (MTNR1A) is polymorphic in buffaloes. Single nucleotide polymorphisms (SNPs) in gene MTNR1A have been associated with fertility in female buffalo. The knowledge and tools are available to lift the reproductive performance of buffalo. This is highly important as the global demand for nutritious buffalo food products has undergone a sharp rise, and continues to grow. Buffalo can make an important contribution to affordable, nutritious animal protein. This will help address global nutritional security. • Buffalo are important livestock in global food security and in the livelihood of smallholder farmers. • Seasonal breeding in buffalo is determined by exogenous (photoperiod, climate, nutrition, management) and endogenous (hormones, genotype) factors. • Melatonin influences seasonal reproduction through photoperiodic time measurement in the brain and by direct actions at the ovaries and testes. [ABSTRACT FROM AUTHOR]

The study aimed to verify the influence of different milking machine setting parameters on Italian Mediterranean buffalo milking performance. Data on milking machine settings (Milking Dry Tests [MDTs]) and milk Test-day (TD) were collected by the Italian Breeders Association (AIA). Data for each TD consisted of milk yield (MY), fat percentage (FP), protein percentage (PP), lactose percentage (LP) and linear score (LS). Working vacuum level (VL), pulsation ratio (PR), automatic cluster removal system (AC) and effective vacuum reserve (EVR) were recorded from each MDT. A total of 558 MDTs, 217,967 TD, collected from 43,593 buffaloes in 198 buffalo farms were utilised. MDT, TD and Animal data (AD) were analysed through a mixed linear model and a logistic regression model was used to evaluate the relationship between VL and EVR. Analysis of MY and quality revealed that an incorrect setting of the milking was responsible for a higher LS and lower MY and LP, along with higher FP and PP compared to farms with adequate EVR. Except for PP, the height of the milking system significantly influenced all milk parameters. Conversely, VL affected all milking traits. A higher PR (70:30) was responsible for significantly lower LS and higher FP compared to a 60:40 PR. Similarly, the presence of AC showed a significant effect on FP and MY and a slight reduction in LS. Finally, the diameter and length of the pipeline (DL) ratio lower than 2.5 m was associated with a decrease in LS. These results suggest that buffaloes require specific milking parameters. Buffalo has a specific requirement for milking parameters. Incorrect milking setting increase somatic cells and reduce lactose content. Vacuum level (VL) affects milk yield (MY) and quality as well as somatic cells and lactose. [ABSTRACT FROM AUTHOR]

Background: Several studies show that natural foods are a source of compounds with anticancer properties that affect the gut microbiota and its metabolites. In the present study, we investigate the effect of a delactosed buffalo milk whey by-product (DMW) on colorectal carcinogenesis. Methods: The effect of DMW on colorectal carcinoma (CRC) was investigated in the established mouse model of azoxymethane (AOM)-induced colon carcinoma, which closely resembles the human clinical condition of CRC. The effect of DMW on CRC immortalized cell lines was also evaluated to further identify the antineoplastic mechanism of action. Results: Pretreatment of AOM-treated mice with DMW significantly (P < 0.05) reduced the percentage of mice bearing both aberrant crypt foci with more than four crypts (which are early precancerous lesions that progress to CRC) and tumors. In addition, DMW completely counteracted the effect of AOM on protein expression of caspase-9, cleaved caspase-3 and poly ADP-ribose polymerase in colonic tissue. Administration of DMW alone (i.e. without AOM) resulted in changes in the composition of the gut microbiota, leading to enrichment or depletion of genera associated with health and disease, respectively. DMW was also able to restore AOM-induced changes in specific genera of the gut microbiota. Specifically, DMW reduced the genera Atopobiaceae, Ruminococcus 1 and Lachnospiraceae XPB1014 and increased the genera Parabacteroides and Candidatus Saccharimonas, which were increased and reduced, respectively, by AOM. Blood levels of butyric acid and cancer diagnostic markers (5-methylcytidine and glycerophosphocholine), which were increased by AOM treatment, were reduced by DMW. Furthermore, DMW exerted cytotoxic effects on two human CRC cell lines (HCT116 and HT29) and these effects were associated with the induction of apoptotic signaling. Conclusions: Our results suggest that DMW exerts chemopreventive effects and restores the gut microbiota in AOM-induced CRC, and induces cytotoxic effect on CRC cells. DMW could be an important dietary supplement to support a healthy gut microbiota and reduce the prevalence of CRC in humans. ATYCseWUm9Z2DGDMVykgy6 Video Abstract [ABSTRACT FROM AUTHOR]

Simple Summary: Goats are susceptible to ectoparasite infection, and lice control is based on studies conducted in cattle and sheep through the topical application of several synthetic insecticides such as organophosphates, carbamates, and pyrethroids. However, the overuse of these compounds has led to insect resistance phenomena. Thus, natural insecticides could be preferred, considering that synthetic insecticides and their metabolites negatively impact the environment and human and animal health. The aim of the present study was to evaluate the efficacy and safety of neem oil on caprine pediculosis and on kids' growth performances. The neem (Azadirachta indica) belongs to the Meliaceae family, and in Eastern countries it is mainly considered for the insecticidal activities of the kernel oil. The neem seeds contain bioactive principles, such as azadirachtin A, salannin, nimbin, and nimbolide. The trial was carried out on 24 kids, 120 days old, maintained in open yards. Animals were divided in 4 homogeneous groups (n = 6 animals/group) based on age, louse count, body condition score (BCS) and live body weight: Control Group (C, saline NaCl, 0.9%), Neem Group 1 (NO-100, 100 mL of neem oil per 10 kg), Neem Group 2 (NO-200, 200 mL/10 kg), Neem Group 3 (NO-300, 300 mL/10 kg). The treatments were performed by spraying the insecticide on the goat's body. The study lasted 56 days, and weekly, the kids underwent louse count, BCS and body weight determination, and FAMACHA score. Data were analyzed by ANOVA for repeated measures. The species of lice identified was Linognathus stenopsis. Kids belonging to NO-200 and NO-300 showed a stronger reduction of louse count throughout the study (>95%). The daily weight gain recorded was significantly higher (p < 0.05) in NO-300 than C. No differences were found for BCS and FAMACHA scores. The results of this trial showed that the administration of neem oil to control caprine pediculosis caused by sucking lice represents an alternative to synthetic compounds. [ABSTRACT FROM AUTHOR]

Background: Green feed diet in ruminants exerts a beneficial effect on rumen metabolism and enhances the content of milk nutraceutical quality. At present, a comprehensive analysis focused on the identification of genes, and therefore, biological processes modulated by the green feed in buffalo rumen has never been reported. We performed RNA-sequencing in the rumen of buffaloes fed a total mixed ration (TMR) + the inclusion of 30% of ryegrass green feed (treated) or TMR (control), and identified differentially expressed genes (DEGs) using EdgeR and NOISeq tools. Results: We found 155 DEGs using EdgeR (p-values < 0.05) and 61 DEGs using NOISeq (prob ≥0.8), 30 of which are shared. The rt-qPCR validation suggested a higher reliability of EdgeR results as compared with NOISeq data, in our biological context. Gene Ontology analysis of DEGs identified using EdgeR revealed that green feed modulates biological processes relevant for the rumen physiology and, then, health and well-being of buffaloes, such as lipid metabolism, response to the oxidative stress, immune response, and muscle structure and function. Accordingly, we found: (i) up-regulation of HSD17B13, LOC102410803 (or PSAT1) and HYKK, and down-regulation of CDO1, SELENBP1 and PEMT, encoding factors involved in energy, lipid and amino acid metabolism; (ii) enhanced expression of SIM2 and TRIM14, whose products are implicated in the immune response and defense against infections, and reduced expression of LOC112585166 (or SAAL1), ROR2, SMOC2, and S100A11, encoding pro-inflammatory factors; (iii) up-regulation of NUDT18, DNAJA4 and HSF4, whose products counteract stressful conditions, and down-regulation of LOC102396388 (or UGT1A9) and LOC102413340 (or MRP4/ABCC4), encoding detoxifying factors; (iv) increased expression of KCNK10, CACNG4, and ATP2B4, encoding proteins modulating Ca2+ homeostasis, and reduced expression of the cytoskeleton-related MYH11 and DES. Conclusion: Although statistically unpowered, this study suggests that green feed modulates the expression of genes involved in biological processes relevant for rumen functionality and physiology, and thus, for welfare and quality production in Italian Mediterranean dairy buffaloes. These findings, that need to be further confirmed through the validation of additional DEGs, allow to speculate a role of green feed in the production of nutraceutical molecules, whose levels might be enhanced also in milk. [ABSTRACT FROM AUTHOR]

One animal-based method to evaluate welfare is the presence of stress. In dairy ruminants, the responses to stressors include the activation of both neuroendocrine and autonomous nervous system that can be evaluated through an endocrine assessment. The present study aimed to validate a radioimmunoassay method for cortisol in buffalo milk. Three formulations (whole and skimmed milk and whey) and three solvents (methanol diethyl ether and dichloromethane) were tested: methanol was characterised by the best extraction efficiency (69.88%), whey cortisol concentrations showed a significant correlation with whole extracted milk and were not affected by fat content variation during the milking session. The RIA used in the present study showed good precision, sensitivity and specificity: the dilutions test indicated the high reproducibility of the results, overlapping of the dilution curve and standard curve highlighted high specificity and the lack of interfering factors by buffalo whey matrix. It is concluded that the present assay suits the cortisol measurement in buffalo milk and the ranges described could be employed in the calibration of a biosensing technologies directly integrated in milking parlour systems. Buffalo milk whey revealed to be a matrix of great interest because of its high stability in terms of storage, transportation and processing. RIA method suits the cortisol measurement in buffalo milk Ranges described can be employed in the calibration of biosensors for non-invasive assessment of cortisol [ABSTRACT FROM AUTHOR]

The temperature–humidity index (THI) has been commonly used to analyse heat stress in dairy cattle, but little is known about its effects on buffaloes. In this study, daily milk yield (MY), fat percentage (FP), protein percentage (PP) and somatic cell count (SCC) data from 808 buffalo cows plus environmental temperature and relative humidity were used to investigate the consequence of heat stress. Two mixed models were used to evaluate the impact of THI on MY, FP, PP and log transformed SCC (SCS). The effect of THI was significant for PP, FP and SCS, whereas its interaction with parity was statistically significant for PP and SCS. The relationship between PP and FP and THI was positive but of different magnitude according to the parity. When THI was below 62, an unfavourable effect was observed, especially in primiparous buffalo cows. A significant interaction between SCS and THI across parities was also observed. The effect of THI on MY across parities was not definite but overall a favourable relationship was observed. Our findings depict a susceptibility of buffaloes to low values of THI, suggesting an optimal THI range for water buffaloes between 59 and 63, although some deleterious effects were observed in primiparous buffaloes at THI values lower than 62. Additional investigations are needed to better elucidate the influence of THI on buffalo species. The overall effect of THI on buffalo diverges from what commonly observed in dairy cattle Cold stress affects milk and udder health in buffaloes The effect of THI on buffaloes' performance depends on parity, with a larger susceptibility in primiparous than pluriparous buffalo cows Udder health in buffaloes, evaluated using somatic cell count, is also affected by THI [ABSTRACT FROM AUTHOR]

Simple Summary: Mastitis can be considered one of the costliest diseases of the dairy Mediterranean Buffalo. Early detection of the disease is thus of great importance to farmers, to reduce or prevent production losses. The measurement of electrical conductivity (EC) of milk is a relatively simple and inexpensive technique, and it has been studied as a routine method for the diagnosis of mastitis in dairy farm. Limited information is currently available on the relationships among EC, production traits and somatic cells count in Italian Mediterranean Buffalo. Hence, the aim of this study was to investigate those correlations using data collected at a commercial Italian buffalo farm. We can conclude that in buffalo, as in other species, there is a strong relationship between EC and somatic cells. Furthermore, we observed that, if the objective is to have an additional and informative parameter for early detection of mastitis, frequent EC recording, possibly performed over a longer period of time, is more effective than recording EC only a few times. Even if our results are encouraging, further studies are needed, in order to validate them, especially if the objective is the development of udder disease prediction models. The measurement of milk electrical conductivity (EC) is a relatively simple and inexpensive technique that has been evaluated as a routine method for the diagnosis of mastitis in dairy farms. The aim of this study was to obtain further knowledge on relationships between EC, production traits and somatic cell count (SCC) in Italian Mediterranean Buffalo. The original dataset included 5411 records collected from 808 buffalo cows. Two mixed models were used to evaluate both the effect of EC on MY, PP and FP and EC at test-day, and the effect of EC on somatic cell score (SCS) by using five different parameters (EC_param), namely: EC collected at the official milk recording test day (EC_day0), EC collected 3 days before official milk recording (EC_day3), and three statistics calculated from EC collected 1, 3 and 5 days before each test-day, respectively. All effects included in the model were significant for all traits, with the only exception of the effect of EC nested within parity for FP. The relationship between EC and SCS was always positive, but of different magnitude according to the parity. The regression of EC on SCS at test-day using different EC parameters was always significant except when the regression parameter was the slope obtained from a linear regression of EC collected over the 5-day period. Moreover, in order to evaluate how well the different models fit the data, three parameters were used: the Average Information Criteria (AIC), the marginal R2 and the conditional R2. According to AIC and to both the Marginal and Conditional R2, the best results were obtained when the regression parameter was the mean EC estimated over the 5-day period. [ABSTRACT FROM AUTHOR]

Recent pharmacological research on milk whey, a byproduct of the dairy industry, has identified several therapeutic properties that could be exploited in modern medicine. In the present study, we investigated the anticancer effects of whey from Mediterranean buffalo (Bubalus bubalis) milk. The antitumour effect of delactosed milk whey (DMW) was evaluated using the HCT116 xenograft mouse model of colorectal cancer (CRC). There were no discernible differences in tumour growth between treated and untreated groups. Nevertheless, haematoxylin and eosin staining of the xenograft tissues showed clearer signs of different cell death in DMW-treated mice compared to vehicle-treated mice. Detailed biochemical and molecular biological analyses revealed that DMW was able to downregulate the protein expression levels of c-myc, phospho-Histone H3 (ser 10) and p-ERK. Moreover, DMW also activated RIPK1, RIPK3, and MLKL axis in tumour tissues from xenograft mice, thus, suggesting a necroptotic effect. The necroptotic pathway was accompanied by activation of the apoptotic pathway as revealed by increased expression of both cleaved caspase-3 and PARP-1. At the molecular level, DMW-induced cell death was also associated with (i) upregulation of SIRT3, SIRT6, and PPAR-γ and (ii) downregulation of LDHA and PPAR-α. Overall, our results unveil the potential of whey as a source of biomolecules of food origin in the clinical setting of novel strategies for the treatment of CRC. [ABSTRACT FROM AUTHOR]

Abstract: A comprehensive study of the efficiency of synchronization of ovulation and timed artificial insemination (TAI) was undertaken in a large group of Italian Mediterranean buffaloes at a commercial dairy. A total of 2791 synchronization protocols were carried out on 857 animals over 3 years. Of these protocols, 823 (29.5%) did not proceed beyond Day 7 (due to the absence of a vascularized CL) and 620 (22.2%) were discontinued on Day 10 (due to the absence of follicles >1.0 cm and tonic uteri); hence, 1443 (51.7%) protocols did not progress to TAI. Data were analyzed for four periods: P1, transition to spring (from breeding season to low breeding season); P2, low breeding season; P3, transition to fall (low breeding season to breeding season); and P4, breeding season. No differences were found among the four periods in terms of the proportion of protocols that did not result in TAI. Of the 857 buffaloes, 660 (77%) conceived and delivered a calf. The average number of TAI per pregnancy was 2.1 and ranged from 1.9 to 2.3 across years. Logistic regression analysis showed that buffaloes that calved during P3 had a higher odds ratio for pregnancy (1.380; P < 0.05) than buffaloes that calved in other periods. Pregnancy was also influenced by the calving to service period (odds ratio = 0.977; P < 0.01) and the pregnancy per AI (P/AI) at successive TAI (odds ratio = 1.480; P < 0.01). The pregnancy per AI at the first TAI (424/857, 49.5%) was greater (P < 0.01) than in subsequent TAI. The occurrence of late embryonic mortality (between Days 27 and 45 after TAI) was similar among the four periods. These findings indicated that there are distinct seasonal differences in the response of Italian Mediterranean buffaloes to synchronization and TAI. [Copyright &y& Elsevier]

The transcriptome profiles were compared for buffalo embryos with normal growth and embryos with retarded growth on Day 25 after mating. Embryos with retarded growth on Day 25 after mating have a reduced likelihood of undergoing attachment to the uterine endometrium and establishing a pregnancy. Italian Mediterranean buffaloes were mated by AI and on Day 25 underwent trans-rectal ultrasonography to ascertain embryo development. Embryos with an embryonic width (EW)>2.7 mm were classed as normal embryos and embryos with an EW<2.7 mm were classed as retarded embryos. Three buffaloes with embryos of the largest EW (3.7, 3.7 and 3.9 mm) and three buffaloes with embryos of the smallest EW (1.5, 1.6 and 1.9 mm) were slaughtered on Day 27 to recover embryos for transcriptome analysis using a bovine custom designed oligo array. A total of 1,047 transcripts were differentially expressed between embryos with normal growth and embryos with retarded growth. Retarded embryos showed 773/1,047 (74%) transcripts that were down-regulated and 274/1,047 (26%) transcripts that were up-regulated relative to normal embryos; in silico analyses focused on 680/1,047 (65%) of the differentially expressed transcripts. The most altered transcripts observed in retarded embryos were associated with membrane structure and function and with metabolic and homeostasis maintenance functions. Other notable functions altered in retarded embryos were developmental processes and in particular nervous system differentiation and function. Specific biochemical pathways such as the complement cascade and coagulation were also altered in retarded embryos. It was concluded from the findings that buffalo embryos with retarded growth on Day 25 after mating show altered gene expression compared with normal embryos, and some de-regulated functions are associated with attachment to the uterine endometrium. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to evaluate the effect of osteopontin (OPN), an ubiquitous acid glycoprotein, on in vitro sperm capacitation and on in vitro embryo production (IVEP) efficiency in buffalo. In experiment 1, after swim-up separation the sperm were incubated in Tyrode albumin lactate pyruvate medium in the absence of capacitating agents (control), with the standard concentration of heparin (0.01 mM) and three different concentrations of OPN (0.1, 1, and 10 mcg/mL), both in the presence and absence of heparin, for 2 and 4 hours. Capacitation was assessed indirectly by estimating the percentage of acrosome-reacted sperm after incubation with lysophosphatidylcholine. In order to determine the effect of OPN, in the presence of heparin, on fertilization (Experiment 2) and in vitro embryo development (experiment 3), in vitro–matured buffalo oocytes were fertilized in the presence of 0, 0.1, 1, and 10 mcg/mL of OPN. After IVF, the presumptive zygotes were dezonated, fixed, stained, and then evaluated microscopically. At Days 5 and 7 of culture, the cleavage and blastocyst rates were evaluated, respectively. Two hours of treatment with OPN at the two higher concentrations (1 and 10 mcg/mL) promoted in vitro capacitation of buffalo sperm (experiment 1). A synergic action of OPN with heparin was also done for all OPN concentrations tested. At 4 hours incubation, all treatments, including heparin (20.4%), improved (P < 0.01) capacitation compared with the control (16.2%). Interestingly, the best results were reported in all groups treated with OPN + heparin (40.8%, 38.6%, and 33.8%, respectively; P < 0.01). The addition of OPN to the IVF medium had a positive influence on total penetration, synchronous pronuclei formation (experiment 2), and IVEP efficiency (experiment 3). In particular, the two lower concentrations of OPN (0.1 and 1 mcg/mL), compared with the control, gave higher synchronous pronuclei formation (73.5%, 75.0%, and 46.5%, respectively; P < 0.01) and cleavage rates (70.3%, 71.6%, and 59.3%, respectively; P < 0.01). Interestingly, the treatments also improved blastocyst yields (29.3%, 30.3%, and 19.4%, respectively; P < 0.01). In conclusion, these results indicate that adding OPN to the IVF system improves IVEP efficiency by enhancing in vitro sperm capacitation and blastocyst yields in buffalo. [Copyright &y& Elsevier]

Abstract: The use of sexed semen technology in buffaloes is nowadays becoming more and more accepted by farmers, to overcome the burden of unwanted male calves with related costs and to more efficiently improve production and genetic gain. The aim of this study was to verify the coupling of some variables on the efficiency of pregnancy outcome after deposition of sexed semen through AI. Pluriparous buffaloes from two different farms (N = 152) were screened, selected, and subjected to Ovsynch protocol for AI using nonsexed and sexed semen from four tested bulls. AI was performed in two distinct periods of the year: September to October and January to February. Neither farms nor bulls had a significant effect on pregnancy rates pooled from the two periods. The process for sexing sperm cells did not affect pregnancy rates at 28 days after AI, for nonsexed and sexed semen, respectively 44/73 (60.2%) and 50/79 (63.2%), P = 0.70, and at 45 days after AI, for nonsexed and sexed semen, respectively 33/73 (45.2%) and 33/79 (49.3%), P = 0.60. Pregnancy rate at 28 days after AI during the transitional period of January to February was higher when compared with September to October, respectively 47/67 (70.1%) versus 47/85 (55.2%), P = 0.06. When the same pregnant animals were checked at Day 45 after AI, the difference disappeared between the two periods, because of a higher embryonic mortality, respectively 32/67 (47.7%) versus 40/85 (47.0%), P = 0.93. Hematic progesterone concentration at Day 10 after AI did not distinguish animals pregnant at Day 28 that would or would not maintain pregnancy until Day 45 (P = 0.21). On the contrary, when blood samples were taken at Day 20 after AI, the difference in progesterone concentration between pregnant animals that would maintain their pregnancy until Day 45 was significant for both pooled (P = 0.00) and nonsexed (P = 0.00) and sexed semen (P = 0.09). A similar trend was reported when blood samples were taken at Day 25, being highly significant for pooled, nonsexed, and sexed semen (P = 0.00). Hematic progesterone concentration between the two periods of the year was highly significant for pregnant animals at 28 days from AI when blood samples were taken at Day 20 after AI for pooled, nonsexed, and sexed semen, respectively P = 0.00, 0.00, and 0.06, and for pregnant animals at Day 45 for pooled, nonsexed, and sexed semen, respectively P = 0.00, 0.00, and 0.01. From these results, it can be stated that hematic progesterone concentration measurement since Day 20 after AI can be predictive of possible pregnancy maintenance until Day 45. Furthermore, the transitional period of January to February, although characterized by a higher pregnancy outcome when compared with September to October, suffers from a higher late embryonic mortality as evidenced by a significant different hematic progesterone concentration between the two periods at Day 20 after AI. [Copyright &y& Elsevier]

It was demonstrated that cysteamine supplementation during in vitro maturation (IVM) improves embryo development by increasing glutathione (GSH) synthesis in several species. An improved developmental competence of oocytes matured in the presence of cysteamine was also recorded in buffalo species. The purpose of this work was to investigate (1) if glutathione is de novo synthesized during in vitro maturation of buffalo oocytes, (2) if cysteamine improves buffalo embryo development via an increase in GSH synthesis, and (3) if the inhibition of glutathione synthesis by buthionine sulfoximide (BSO), in the presence or absence of cysteamine, affects subsequent embryo development and GSH synthesis.Cumulus–oocytes complexes (COCs), recovered from slaughtered animals, were matured in vitro in TCM199+10% fetal calf serum (FCS), 0.5 μg/ml FSH, 5 μg/ml LH and 1 μg/ml 17-β-estradiol in the absence or presence of cysteamine (50 μM), with or without 5 mM BSO. Glutathione content was measured by high-performance liquid chromatography (HPLC) and fluorimetric analysis in immature oocytes and in oocytes matured in the different experimental conditions.In a second experiment, the mature oocytes were in vitro fertilized and cultured for 7 days in order to assess development to blastocysts (BLs). It was demonstrated that buffalo oocytes synthesize glutathione during in vitro maturation and that cysteamine increases glutathione synthesis. Furthermore, the promoting effects of cysteamine on embryo development and GSH synthesis were neutralized by buthionine sulfoximide. These results indicate that glutathione plays a critical role on buffalo embryo development. [Copyright &y& Elsevier]

The aim of this study was to test the feasibility of genomic selection in the Italian Mediterranean water buffalo, which is farmed mainly in the south Italy for milk, and mozzarella, production. A total of 498 animals were genotyped at 49,164 loci. Test day records (80,417) of milk (MY), fat (FY) and protein (PY) yields from 4127 cows, born between 1975 and 2009, were analysed in a three-trait model. Cows born in 2008 and 2009 with phenotypes and genotypes were selected as validation animals (n = 50). Variance components (VC) were estimated with BLUP and ssGBLUP. Heritabilities for BLUP were 0.25 ± 0.02 (MY), 0.16 ± 0.01 (FY) and 0.25 ± 0.01 (PY). Breeding values were computed using BLUP and ssGBLUP, using VC estimated from BLUP. ssGBLUP was applied in five scenarios, each with a different number of genotypes available: (A) bulls (35); (B) validation cows (50); (C) bulls and validation cows (85); (D) all genotyped cows (463); (E) all genotypes (498). Model validation was performed using the LR method: correlation, accuracy, dispersion, and bias statistics were calculated. Average correlations were 0.71 ± 0.02 and 0.82 ± 0.01 for BLUP and ssGBLUP-E, respectively. Accuracies were also higher in ssGBLUP-E (0.75 ± 0.03) compared to BLUP (0.57 ± 0.03). The best dispersions (i.e. closer to 1) were found for ssGBLUP-C. The use of genotypes only for the 35 bulls did not change the validation values compared to BLUP. Results of the present study, even if based on small number of animals, showed that the inclusion of genotypes of females can improve breeding values accuracy in the Italian Buffalo. The genotypes of males did not improve the predictions. Genotypes of females improve breeding values accuracy. Slight increase in prediction accuracy with weighted ssGBLUP. [ABSTRACT FROM AUTHOR]

Native goat breeds play an important role both in the safeguarding of biodiversity and the development of local economy. This study aimed to evaluate milk coagulation properties (MCP) of local (Garganica, Girgentana, Maltese, Mediterranean Red) and cosmopolitan (Saanen) goat breeds. From May to August 2018, 46 goats were sampled monthly in an experimental farm; milk samples were analysed for fat, protein and lactose percentages, pH, SCC and MCP. Data were evaluated through a mixed linear model with repeated measures, including breed, month of lactation and parity as fixed effects. Month of lactation affected all studied traits, and breed affected MCP, protein percentage, lactose percentage and pH. Milk composition and coagulation properties were better in local breeds than Saanen. In particular, milk of local breeds was richer in protein percentage, and coagulated and reached 20 mm of curd firmness on average 3.14 and 2.56 min earlier than Saanen, respectively. Moreover, the firmest curd was observed in Girgentana (31.44 mm) and the weakest in Garganica (21.72 mm). Rennet coagulation time and curd firmness decreased towards the end of lactation, differing significantly between May (10.62 min and 31.69 mm, respectively) and August (8.33 min and 20.69 mm, respectively), whereas curd-firming time only differed between May and June–July. In conclusion, milk of local breeds showed better milk coagulation ability than the cosmopolitan breed. Breed and month of lactation were important sources of variation of goat MCP. Local goat breeds showed better MCP than Saanen. Valorisation of the milk from the local breeds might be a tool to preserve native genetic resources. [ABSTRACT FROM AUTHOR]

The aim of this study is to combine advanced GC-MS and metabolite identification in a robust and repeatable technology platform to characterize the metabolome of buffalo milk and mozzarella cheese. The study utilized eleven dairies located in a protected designation of origin (PDO) region and nine dairies located in non-PDO region in Italy. Samples of raw milk (100 mL) and mozzarella cheese (100 g) were obtained from each dairy. A total of 185 metabolites were consistently detected in both milk and mozzarella cheese. The PLS-DA score plots clearly differentiated PDO and non-PDO milk and mozzarella samples. For milk samples, it was possible to divide metabolites into two classes according to region: those with lower concentrations in PDO samples (galactopyranoside, hydroxybuthyric acid, allose, citric acid) and those with lower concentrations in non-PDO samples (talopyranose, pantothenic acid, mannobiose, etc.,). The same was observed for mozzarella samples with the proportion of some metabolites (talopyranose, 2, 3-dihydroxypropyl icosanoate, etc.,) higher in PDO samples while others (tagatose, lactic acid dimer, ribitol, etc.,) higher in non-PDO samples. The findings establish the utility of GC-MS together with mass spectral libraries as a powerful technology platform to determine the authenticity, and create market protection, for "Mozzarella di Bufala Campana." [ABSTRACT FROM AUTHOR]

δ-Valerobetaine (δVB) is a constitutive milk metabolite with antioxidant and anti-inflammatory activities. Here, we tested the antineoplastic properties of milk δVB on human colorectal cancer cells. CCD 841 CoN (non-tumorigenic), HT-29 (p53 mutant adenocarcinoma) and LoVo (APC/RAS mutant adenocarcinoma) cells were exposed to 3 kDa milk extract, δVB (2 mM) or milk+δVB up to 72 h. Results showed a time- and dose-dependent capability of δVB to inhibit cancer cell viability, with higher potency in LoVo cells. Treatment with milk+δVB arrested cell cycle in G2/M and SubG1 phases by upregulating p21, cyclin A, cyclin B1 and p53 protein expressions. Noteworthy, δVB also increased necrosis (P < 0.01) and when used in combination with milk it improved its activity on live cell reduction (P < 0.05) and necrosis (P < 0.05). δVB-enriched milk activated caspase 3, caspase 9, Bax/Bcl-2 apoptotic pathway and reactive oxygen species (ROS) production, whereas no effects on ROS generation were observed in CCD 841 CoN cells. The altered redox homeostasis induced by milk+δVB was accompanied by upregulation of sirtuin 6 (SIRT6). SIRT6 silencing by small interfering RNA blocked autophagy and apoptosis activated by milk+δVB, unveiling the role of this sirtuin in the ROS-mediated apoptotic LoVo cell death. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the repeat breeding condition in Italian Mediterranean buffaloes that failed to conceive after at least 300 days in milk. The trial was carried out on 40 pluriparous Italian Mediterranean buffaloes with more than 300 days in milk. All the animals underwent ultrasound examination to assess endometrial thickness and oestrus synchronization by the Ovsynch-TAI Program. On the day of oestrus, blood samples were collected for the haemocytometric cell count and biochemical assay, and the animals were slaughtered in a local abattoir. A post-mortem uterine flushing was performed using sterile saline for microbiological analyses. Furthermore, uterine biopsies were carried out for histopathological assessment. Finally, endometrial samples were used for real-time PCR (RT-PCR) analysis to evaluate the expression of genes involved in innate immune recognition of pathogens and the inflammatory response, such as Toll-like receptor (TLR)1, TLR8, interleukin (IL)-1β, IL-6, IL-8, COL4A2, connective tissue growth factor (CTGF), and cysteine-rich angiogenic inducer 61 (CYR61). Statistical analysis was performed by one-way ANOVA. Based on the infiltration of lymphocytes and plasma cells or endometrial gland, lymphatic, and blood vessel ectasia recorded in the histopathological examination, the animals were classified into three groups: healthy (H Group; n = 5), moderate endometritis (M Group; n = 25), and severe endometritis (S Group; n = 10). A significantly greater (P < 0.01) endometrial thickness was recorded in the S Group compared to that in the H and M Group (1.07 ± 0.03 vs. 0.70 ± 0.07 and 0.81 ± 0.04 cm in the S, H, and M Group, respectively). The white blood cell count was lower in the H Group compared to that in the M and S Group (6.3 ± 0.6 vs. 9.3 ± 0.4 and 10.5 ± 0.5 in the H, M, and S Group, respectively). To perform RT-PCR analysis, five animals from groups M and S were randomly selected in order to have balanced results. A higher (P < 0.01) expression of TLR1 , together with a lower expression of COL4A2, IL-1β, IL-6, IL-8, and CYR61 , was recorded in the H Group, compared to both the M and S Groups. In conclusion, about 90% of repeat breeder buffaloes show moderate or severe endometritis, associated with an altered histopathological endometrial profile and altered mRNA expression of pro-inflammatory and fibrotic factors. • The aim of this study was to investigate the repeat breeding condition in buffaloes. • The majority of repeat breeder show moderate or severe subclinical endometritis. • Repeat breeders showed altered pro-inflammatory and fibrotic factors mRNA expression. • Subclinical endometritis might contribute to the reduced fertility in buffaloes. [ABSTRACT FROM AUTHOR]

The aim of the present study was to determine if space allocation influenced the concentration of biomolecules in buffalo milk and dairy products. Intensively housed buffaloes (n = 96) were randomly assigned to 2 groups according to days in milk, parity, and milk yield: group S10 had a space allocation of 10 m2 per buffalo and group S15 had a space allocation of 15 m2 per buffalo. Individual milk yield was recorded daily. Twice a month, a bulk milk sample was collected for each group, as well as whey, ricotta, and mozzarella cheese, to assess cheese yield and to conduct HPLC-electrospray ionization-tandem mass spectrometry, milk antioxidant activity, and cell viability analyses. We tested milk extracts from the 2 groups in vitro to evaluate their efficacy in counteracting endothelial oxidative damage induced by high glucose. We evaluated reproductive function in 28 buffaloes from each group using the Ovsynch-timed artificial insemination program. We observed no differences in milk quantity or quality in terms of fat, protein, or lactose, and reproductive function did not differ between the 2 groups. Compared with group S10, group S15 had higher concentrations of carnitine (56.7 ± 1.1 vs. 39.8 ± 0.7 mg/L in milk and 40.9 ± 0.8 vs. 31.7 ± 0.7 mg/L in whey), acetyl- l -carnitine (51.9 ± 0.3 vs. 39.7 ± 0.7 mg/L in milk and 41.1 ± 1.7 vs. 28.7 ± 2.6 mg/L in whey), propionyl- l -carnitine (34.8 ± 1.0 vs. 21.0 ± 0.9 mg/L in milk and 26.9 ± 0.8 vs. 17.6 ± 1.2 mg/L in whey), glycine betaine (23.1 ± 1.9 vs. 13.5 ± 1.6 mg/L in milk and 10.7 ± 0.4 vs. 7.9 ± 0.5 mg/L in whey), and δ-valerobetaine (24.2 ± 0.5 vs. 16.7 ± 0.5 mg/L in milk and 22.0 ± 0.9 vs. 15.5 ± 0.7 mg/L in whey). Group S15 also had higher total antioxidant activity than group S10 (56.7 ± 1.9 vs. 46.4 ± 1.13 m M Trolox equivalents). Co-incubation of high-glucose-treated endothelial cells with milk extracts from group S15 improved cell viability compared with cells treated with high glucose only; it also reduced intracellular lipid peroxidation (144.3 ± 0.4 vs. 177.5 ± 1.9%), reactive oxygen species (141.3 ± 0.9 vs. 189.3 ± 4.7 optical density units), and cytokine release (tumor necrosis factor-α, IL-1β, IL-6). Greater space allocation was associated with higher levels of biomolecules in buffalo milk. This could have been the result of improved welfare in buffaloes that were allocated more space. [ABSTRACT FROM AUTHOR]

Studies on goat milk have mainly focused on cosmopolitan breeds and very limited information is available on local breeds, which is important for biodiversity preservation and local cheese production. The aim of this study was to evaluate the breed effect on milk yield, composition and somatic cell score (SCS) of five local Italian goat breeds (Garganica, Girgentana, Jonica, Maltese and Mediterranean Red) compared with a cosmopolitan specialized dairy breed (Saanen). A total of 60 goats (10 per breed) from an experimental farm were enrolled in the study. Milk yield, composition and SCS were recorded and analyzed every 2 weeks during the entire lactation. Data were analyzed using a mixed model with repeated measures. Saanen yielded between 0.27 and 0.62 kg day -1 more milk than the local breeds. Among local breeds, Maltese and Jonica were the most productive, with an average of 1.28 and 1.25 kg day -1 , respectively, while Mediterranean Red, Garganica and Girgentana produced ≤1 kg day -1. Saanen had the highest SCS (6.81) and the lowest fat content (3.26 %). In relation to protein, Garganica showed the greatest content (3.71 %), and Saanen had a similar content to other local breeds (3.42 %) except for Maltese, which was lower (3.11 %). Saanen and Garganica had the lowest lactose percentage (4.28 % and 4.26 %, respectively). All breeds followed a similar pattern across lactation: SCS and fat and protein content peaked at the end of the lactation, whereas lactose percentage was highest at the beginning of the lactation. Differences between Saanen and the local breeds for milk yield, composition and SCS were consistent across lactation. In conclusion, local breeds produced less milk but with lower SCS and greater fat and lactose content than the Saanen cosmopolitan breed, suggesting a better milk quality. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine whether buffalo heifers maintained at a constant live weight (LW) and body condition score (BCS) by a restricted diet show changes in reproductive function in response to changes in day length. Heifers were fed a low energy diet (86% daily intake recommended for the species) throughout the study, which involved an 8-week period of decreasing day length and a 7-week period of increasing day length. Weekly, LW, BCS and ovarian follicular/oocyte population were assessed. Blood and follicular fluid samples were taken at the time of ovum pick-up for hormone analyses and at 30-day intervals to measure metabolic substrates. Buffalo heifers maintained LW and BCS throughout the study and blood concentrations of the main metabolic hormones, such as GH, insulin, IGF-1 and thyroid hormones were not influenced by photoperiod. Likewise, there were no differences in metabolic substrates such as glucose, NEFA, triglycerides and HDL cholesterol, suggesting that the metabolic condition remained essentially the same. During increasing day length periods, the number of total follicles (8.4 ± 0.5 vs. 11.9 ± 0.6; p <.01), cumulus-oocyte complexes (2.2 ± 0.3 vs 4.3 ± 0.5; p <.01) and superior quality Grade A + B COCs (0.2 ± 0.1 vs 1.1 ± 0.2; p <.01) decreased. These features were associated to reduced oestradiol levels both in plasma (3.9 ± 0.4 vs 9.2 ± 0.5 pg/mL; p <.01) and follicular fluid (49.7 ± 12.9 vs. 109.1 ± 25.8 ng/mL; p =.05), as well as to reduced intrafollicular IGF-1 levels (45.5 ± 4.0 vs. 58.5 ± 3.9 ng/mL; p <.05). These findings suggest that the ovarian function in buffalo heifers is influenced by photoperiod independent of nutritional and metabolic status. Metabolic profile, LW and BCS do not change in buffalo heifers on a restricted diet Photoperiod affects ovarian function in heifers regardless of metabolic status Follicular and oocyte population decrease in increasing day length periods Oestradiol and IGF-1 levels in follicular fluid are affected by photoperiod [ABSTRACT FROM AUTHOR]

Abstract The aim of this work was to evaluate factors affecting ovum capture in superovulated buffaloes, by comparing the morphological features of pre-ovulatory follicles and oocytes, the intrafollicular and plasmatic steroid profile, as well as the expression of genes involved in cumulus expansion and steroid cascade in granulosa cells (GCs) and that of genes involved in contraction-relaxation of the oviduct between superovulated and synchronized buffaloes. Italian Mediterranean Buffalo cows were either synchronized by Ovsynch (n = 25) and superovulated (n = 10) with conventional FSH protocol and sacrificed 18 h after last GnRH. Antral follicular count, recovery rate and oocyte quality were recorded, and plasma and follicular fluid were collected for steroid profile determination. In addition, in 10 animals (5/group), GCs were collected to analyse the mRNA expression of gonadotropin receptors (LHR and FSHR) and genes involved in steroid synthesis, as the cytochrome P450 family 19 (CYP19A1) and the steroidogenic acute regulatory protein (STAR). Moreover, oviducts were collected to evaluate the mRNA expression of estrogen receptor 1 (ER1) and the progesterone receptor (PGR), the vascular endothelial growth factor (VEGF) and the VEGF receptors, i.e. the kinase insert domain receptor (FLK1) and the fms related tyrosine kinase 1 (FLT1). No differences were recorded in steroids plasma concentration between synchronized and superovulated animals whereas intrafollicular E 2 and P 4 concentrations decreased in superovulated group (63.2 ± 10.6 vs 30.3 ± 5.9 ng/mL of E 2 and 130.1 ± 19.8 vs 71.6 ± 8.5 ng/mL of P 4 , respectively in synchronized and superovulated animals; P < 0.05). Interestingly, both the recovery rate (85.7% vs 56.6%, respectively in synchronized and in superovulated animals; P < 0.05) and the percentage of oocytes exhibiting proper cumulus expansion (75% vs 28.1%, respectively in synchronized and in superovulated animals; P < 0.01) decreased in superovulated animals. In addition, the expression of FSHR and CYP19A1 increased while the expression of STAR in GCs decreased (P < 0.05). Finally, in superovulated buffaloes a decreased expression of PGR , ER1 , VEGF and its receptor FLK1 in the oviduct was observed. The results suggest that the exogenous FSH treatment impairs steroidogenesis, affecting both the oviduct and the ovarian function, accounting for the failure of ovum capture in superovulated buffaloes. Highlights • Poor embryo recovery in superovulated buffalo is due to failure in ovum capture. • MOET impairs stereidogenesis and in turn ovarian and oviduct function in buffalo. • Exogenous FSH treatment affects gene expression in granulosa cells and oviduct. [ABSTRACT FROM AUTHOR]

Quaternary ammonium compounds containing N -trimethylamino moiety, such as choline derivatives and carnitine, abundant in meat and dairy products, are metabolic precursors of trimethylamine (TMA). A similar fate is reported for N ε -trimethyllysine and γ-butyrobetaine. With the aim at investigating the metabolic profile of such metabolites in most employed animal dietary sources, HPLC-ESI-MS/MS analyses on ruminant and non-ruminant milk and meat were performed. Results demonstrate, for the first time, the presence of δ-valerobetaine, occurring at levels higher than γ-butyrobetaine in all ruminant samples compared to non-ruminants. Demonstration of δ-valerobetaine metabolic origin, surprisingly, showed that it originates from rumen through the transformation of dietary N ε -trimethyllysine. These results highlight our previous findings showing the ubiquity of free N ε -trimethyllysine in vegetable kingdom. Furthermore, δ-valerobetaine, similarly to γ-butyrobetaine, can be degraded by host gut microbiota producing TMA, precursor of the proatherogenic trimethylamine N-oxide (TMAO), unveiling its possible role in the biosynthetic route of TMAO. [ABSTRACT FROM AUTHOR]

Dairy donkey milking procedures require separating foals from their dams for a few hours a day. Artificial suckling in this species is a good technique for improving milk production and foal welfare. The aim of the work is to compare the effect of two different diets on donkey foals when separated from jennies for milking procedures with and without a milk replacer. Forty newborn Martina Franca donkey foals were subdivided into two experimental groups. Both groups were separated from their respective dams from 8.00to 20.00to allow the jennies to be milked. During the separation, all the foals had access ad libitum to water, hay and feed. During the separation period, one group had the availability of a mechanical milk replacer dispenser, so foals were partially artificially suckled (AS), while the other group had no milk replacer available, and so were totally naturally suckled (NS). The AS group had milk replacer availability until 120±7d of life. Both groups were naturally weaned at 168±7d. Blood samples were collected weekly starting from birth until two wks after weaning (i.e. at 182d), from all the foals included in the trial. Almost all the analytes were influenced by suckling technique and age of foals. Alanine-aminotransferase, aspartate-aminotransferase, alkaline phosphatase, NEFA, lipid hydroperoxides, serum proteins showed the greatest differences between the two experimental groups. Separating foals from their dams for 12hdaily for 24 weeks does not lead to pathological subclinical and metabolic conditions, thus confirming the high rusticity and resistance of the donkey. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the effect of cholesterol-loaded cyclodextrins (CLC) on motility, viability, capacitation status, and in vivo fertility of buffalo frozen-thawed sperm. After the initial semen assessment, buffalo sperm were diluted in BULLXcell extender containing 0- (control), 1.5-, and 3-mg/mL CLC and cryopreserved. At thawing, sperm motility was evaluated by phase contrast microscopy, and viability-capacitation status was assessed by Hoechst 33258-chlortetracycline (CTC) assay. Capacitation status was also evaluated by an indirect immunofluorescence assay to localize phosphotyrosine-containing proteins. Moreover, buffaloes were artificial inseminated to assess the in vivo –fertilizing potential of CLC-treated semen. No differences among control, 1.5-, and 3-mg/mL CLC-treated groups were recorded in both sperm motility (66.5 ± 5.6, 68.8 ± 4.8, and 68.8 ± 4.8, respectively) and viability (86.5 ± 1.9, 87.6 ± 1.5, 88.4 ± 2.3, respectively). However, the extender supplementation with CLC significantly reduced sperm cryocapacitation. Indeed, CLC treatment decreased (P < 0.01) the proportion of sperm showing the CTC pattern B (capacitated sperm) compared with the control (69.6 ± 3.4, 37.8 ± 1.5, and 51.3 ± 4.7, respectively, with 0, 1.5-, and 3-mg/mL CLC; P < 0.01). Furthermore, the percentage of sperm displaying tyrosine-phosphorylated pattern EA (i.e. high capacitation level) was reduced (P < 0.01) in both CLC-treated groups (10.8 ± 3.3 and 5.6 ± 1.6, respectively, with 1.5- and 3-mg/mL CLC) compared with the control (37.3 ± 6.9), reaching values similar to those recorded in fresh semen (11.0 ± 3.5). In addition, treating sperm with 3-mg/mL CLC increased (P < 0.01) the percentage of nonfluorescent (pattern NF), i.e., non-capacitated sperm (41.8 ± 3.6) compared with fresh semen (11.0 ± 6.9). No differences were recorded in pregnancy rates at 60 days post-artificial insemination among control, 1.5- and 3-mg/mL CLC groups (59.7%, 65.6%, and 56.9%, respectively). In conclusion, CLC treatment of buffalo sperm strongly decreases sperm cryocapacitation damages, without affecting the in vivo fertilizing capability. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the effect of resveratrol supplementation of semen extender on fertility parameters of frozen-thawed buffalo sperm. After the initial semen assessment, buffalo semen was cryopreserved in BioXcell containing 0 (control group), 0.5, 1, 10, and 50-μM resveratrol. After thawing, viability, motility, and capacitation status (assessed by localization of phosphotyrosine-containing proteins) were evaluated. Based on the results of the dose-response trial, the concentration of 50 μM was selected for further assessments, such as membrane integrity, total antioxidant capacity, reactive oxygen species, and lipid peroxidation (LPO) levels. Moreover, in vitro fertilizing ability by heterologous IVF and in vivo fertility were assessed. No differences among groups were recorded in sperm motility and viability (on average 52.3 ± 2.1% and 76.6 ± 1.3%, respectively). However, data showed a resveratrol dose-dependent effect on sperm capacitation status, with a significant reduction of the cryopreservation-induced capacitation with the higher concentrations tested. In particular, both 10- and 50-μM resveratrol increased (P < 0.01) the percentage of sperm displaying pattern A (low capacitation level), but treatment with 50-μM resveratrol also decreased (P < 0.01) the proportion of sperm exhibiting pattern EA (high-capacitation level) compared with the control. Interestingly, supplementation of semen extender with resveratrol increased membrane integrity, indicated by the higher percentage of hypo-osmotic swelling positive sperm (55.6 ± 0.6 vs. 48.4 ± 0.7; P < 0.01), and total antioxidant capacity (1.36 ± 0.01 vs. 1.32 ± 0.02 mM/L; P < 0.05) compared with the control. Intracellular reactive oxygen species decreased in resveratrol-treated sperm compared with the control, as indicated by dihydroethidium values (0.17 ± 0.01 and 0.22 ± 0.01 μM/μL dihydroethidium, respectively; P < 0.01). Moreover, when IVF was carried out by using semen treated with 50 μM resveratrol, the normal fertilization rate considerably improved (60.8%, P < 0.05) compared with the control (51.3%). However, no differences were recorded in pregnancy rates at 60 days post-AI with resveratrol-treated semen (50 μM) compared with the control (48.7 vs. 46.5%, respectively). In conclusion, the inclusion of 50-μM resveratrol in the extender decreases capacitation-like changes and oxidative stress, improving membrane stability and in vitro fertilizing ability of buffalo semen. [ABSTRACT FROM AUTHOR]

Recently there has been growing interest in the discovery of new antimicrobial agents to increase safety and shelf-life of food products. Here, we developed an innovative approach by introducing the concept that mitochondrial targeting peptides (MTP) can interact and disrupt bacterial membranes, acting as antimicrobial agents. As proof-of-principle, we used a multidisciplinary strategy by combining in silico predictions, docking simulations and antimicrobial assays, to identify two peptides, MTP1 and MTP2, which were structurally and functionally characterized. Both compounds appeared effective against Listeria monocytogenes , one of the most important foodborne pathogens. Specifically, a significant bactericidal activity was evidenced with EC50 values of 16.8 ± 1.2 μM for MTP1 and 109 ± 7.0 μM for MTP2. Finally, NMR structure determinations suggested that MTP1 would be oriented into the membrane bilayer, while the molecular shape of MTP2 could indicate porin-mediated antimicrobial mechanisms, as predicted using molecular docking analysis. Therefore, MTPs represent alternative sources to design new potential bio-preservatives. [ABSTRACT FROM AUTHOR]

The growing concerns about the relation of livestock activities and sustainability has led to technological progress and the adoption of best practices for reducing their impacts on the environment, including carbon emissions. In southern Italy, farming Mediterranean buffalos for milk production represents a significant portion of the economy. In this study we evaluate the environmental impacts of buffalo milk production using the Life Cycle Assessment approach. The analysis uses a cradle-to-gate system boundaries, an attributional approach and a Functional Unit equal to kg of Energy Corrected buffalo Milk (ECM). We use primary data collected from three farms in Southern Italy, covering a wide range of conditions including organic farming. We investigate the role of allocation method on the overall results and the role of buffalo milk productivity on the environmental impact estimates of milk production. Our results indicate that the environmental impacts of milk production are highly dependent on the chosen allocation method. The life cycle greenhouse gases emission ranges from 1.5 to 2.5 kgCO 2eq per kg of ECM, which are slightly larger compared to those of cow milk; this is mainly due to higher milk productivity of cows compared to buffalos. The hot-spot analysis shows that the largest source of climate change impacts are direct emissions from enteric fermentation. The comparative analysis shows that no farm outperforms the other across the entire spectrum of categories and that the milk productivity of buffalos is a key aspect determining the environmental performance of each farm. [Display omitted] • We use LCA for evaluating the environmental impact of buffalo milk. • Organic and conventional milk production were compared. • Environmental impacts are highly dependent on allocation method. • carbon emissions of buffalo milk are slightly larger than those of cow milk. • Organic farm shows larger impact in land use, water use and non-cancer human health. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate whether the developmental speed affects the cryotolerance of in vitro produced buffalo embryos. In Experiment 1, abattoir-derived oocytes were in vitro matured, fertilized and cultured. The embryos produced by Say 7 of culture were vitrified at the tight morula (TM), early blastocyst (EBL), blastocyst (BL), expanded-blastocyst (XBL) and hatched-blastocyst (HBL) stage. The embryos were vitrified by cryotop in 16.5% ethylene glycol (EG) and 16.5% dimethyl sulfoxide (DMSO) and 0.5 M sucrose. Embryos were warmed in 0.25 M sucrose for 1 min and then in 0.15 M sucrose for 5 min and cultured in vitro for 24 h, to evaluate post-culture viability. In Experiment 2, ovum pick-up (OPU) was carried out on lactating buffaloes to produce embryos that were vitrified- warmed and transferred into synchronized recipients. The lowest (P<0.01) survival rates were recorded with TM (22.4%) and the highest (P<0.01) with HBL (84.5%), whereas intermediate results were observed with EBL, BL and XBL (54.5, 64.7, 67.9%, respectively). Pregnancy rate on both Days 25 and 45 was significantly higher (P<0.05) for faster (XBL and HBL) developing embryos (75.0% and 62.5%, respectively) than for the slower (TM, EBL and BL) developing counterparts (36.4% and 0, respectively). Interestingly, pregnancies to term were only recorded when HBL were transferred (20%). In conclusion, it was demonstrated that the chronology of development is a major factor affecting the cryotolerance of in vitro produced buffalo embryos. [ABSTRACT FROM AUTHOR]

Abstract: This study was designed to evaluate the effect of season on in vivo oocyte recovery and embryo production in Mediterranean Italian buffalo (Bubalus bubalis). For this purpose repeated transvaginal ultrasound-guided ovum pick up (OPU) was conducted twice a week throughout autumn, mid-winter (transitional period) and spring-summer. The number and size of follicles was determined before puncture. The recovered oocytes were first classified in morphological categories and then used for in vitro embryo production (IVEP) according to standard procedures. The mean number of total follicles observed per session did not differ among the three periods we examined (on average 4.6). Although season did not considerably affect the number of oocytes recovered (on average 2.3/buffalo/session), the number of degenerated and abnormally expanded oocytes increased during autumn. Furthermore, the percentage of abnormally expanded oocytes significantly increased during autumn (6.1%) compared with both the transitional period and spring-summer (1.9 and 2.3%, respectively). Interestingly, the embryo output we recorded at day 7, in terms of tight morulae-blastocysts was higher in autumn (30.9%) compared to the other two periods (13.3% and 10.3%, respectively, in spring-summer and in the transitional period; P < 0.01). The results of this trial demonstrated that the morphological features of the oocytes did not vary substantially among the considered periods, with the exception of degenerated and abnormally expanded oocytes. On the other hand, the oocyte developmental competence improved in autumn compared to spring-summer and the transitional period. This datum reflects buffalo reproductive pattern expressed in vivo at Italian latitudes. [Copyright &y& Elsevier]

Abstract: At Italian latitudes, buffalo (Bubalus bubalis) is a seasonally polyestrous species, showing an improved reproductive efficiency when daylight decreases (autumn). The aim of the present study was to evaluate the influence of the season on buffalo oocyte recovery rate, on oocyte quality, assessed on morphological basis, and developmental competence after in vitro fertilization. For this purpose, buffalo ovaries were collected from a local abattoir and the oocytes obtained by aspirating the follicles were evaluated, classified and, if considered of good quality, devolved to the different procedures of IVEP. In general, no differences were found in terms of oocyte recovery per ovary among seasons, but interestingly, the percentage of small oocytes was higher (P <0.05) during spring and summer (0.9±0.1 and 0.9±0.2) compared to autumn and winter (0.3±0.1 and 0.2±0.1). Both cleavage and embryo rate increased during the period from October to December (71.7±3.1 and 26.5±2.1, respectively) compared to the period from April to June (58.0±2.4 and 18.8±1.6, respectively), thus reflecting the in vivo reproductive behavior. Nevertheless, it is worth emphasizing that transferrable embryos were produced in vitro, even during the unfavorable season, but with decreased efficiency. In conclusion, these results suggest to avoid the oocyte collection during spring when planning OPU trials in order to save resources and improve the benefits/costs ratio. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this work was to evaluate whether providing a support of cumulus cells during IVF of buffalo denuded oocytes submitted to vitrification-warming enhances their fertilizing ability. In vitro matured denuded oocytes were vitrified by Cryotop in 20% EG + 20% of DMSO and 0.5 M sucrose and warmed into decreasing concentrations of sucrose (1.25 M–0.3M). Oocytes that survived vitrification were fertilized: 1) in the absence of a somatic support (DOs); 2) in the presence of bovine cumulus cells in suspension (DOs+susp); 3) on a bovine cumulus monolayer (DOs+monol); and 4) with intact bovine COCs in a 1:1 ratio (DOs+COCs). In vitro matured oocytes were fertilized and cultured to the blastocyst stage as a control. An increased cleavage rate was obtained from DOs+COCs (60.9%) compared to DOs, DOs+susp (43.6 and 38.4, respectively; P < 0.01) and DOs+monol (47.5%; P < 0.05). Interestingly, cleavage rate of DOs+COCs was similar to that of fresh control oocytes (67.8%). However, development to blastocysts significantly decreased in all vitrification groups compared to the control (P < 0.01). In conclusion the co-culture with intact COCs during IVF completely restores fertilizing capability of buffalo denuded vitrified oocytes, without improving blastocyst development. [ABSTRACT FROM AUTHOR]