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The aim of this study was to evaluate the influence of temperature during ovary collection/transportation and that of the time interval between ovary collection and processing in the laboratory, on in vitro embryo production efficiency in buffalo species. A retrospective analysis of data collected over the last 4 years in our lab was carried out on 3461 oocytes, recovered over 120 replicates. No differences in oocytes developmental competence were observed in relation to the time interval between ovary collection and processing in the laboratory (3-6 h). On the contrary, a correlation was found between oocyte developmental competence, evaluated in terms of cleavage and blastocyst rate, and temperature during ovary collection/transportation. In particular, lowering temperature during ovary transportation significantly improved both cleavage (74.6 % vs 65.8 and 63.1 % for temperature ranges of 25-30°C, 30-34°C and 34-37°C, respectively ; P

The relationship between variations of weight and BCS was evaluated using growing buffalo heifers, fed diets with different energy level. 12 Murrah bred heifers (age: 790 days, LW: 400 kg), raised in Saint Paul (Brazil) were equally divided in group H and L fed diets with 5.8 UFL/day and 3.6 UFL/day, respectively. At the end of treatment, groups showed significant differences in weight and values of BCS. In this trial each point of BCS, in Murrah heifers, seems to be equivalent to an increase and/or a loss of about 50 kg of live weight. During this trial the mean value of daily weight gain (DWG) was significantly higher in group H (310 g. vs 0 g.; P

The aim of this study was to verify the ratio between real cheese yield at 28 hours and proteins, after values correction according to the regression analysis. Milk characteristics of subjects characterized by a 28CY/protein ratio lower (Group A; n=212) or higher (Group B; n=108) than 56 were analysed by ANOVA in order to evaluate differences between the following milk characteristics: the lactodinamographic parameters (Formagraph, Foss, DN); the complete physic-chemical composition; fat (F), protein (P), casein, lactose, ash, urea, pH and SH; the somatic cells content (SCC) using (Milkoskan); the total aerobic mesophilic flora (TAMF) by the dilution method. Chemical composition of the curd was also determined (protein, fat and ash; ASPA, 1995). The milk protein content was adjusted for the non proteic N content determined in milk as urea (corrected P); the theoretic cheese yield (ThCY) was obtained by the following formula: cheese yield =milk x[-0.88 + 3.50 x P(%)+1.23 x F(%)] x 100 -1 (Altiero et al., 1989) and the ratio between ThCY/FCY and ThCY/28CY were calculated. Other calculated variables were: FCY/Corrected P, 28CY/Corrected P, CDM/Corrected P. Regression analysis was carried out between real cheese yield at 28 hours/proteins ratio and 28CY vs. all the parameters resulted different between the two groups of buffaloes. Real cheese yield at 28 hours/proteins ratio and 28CY were corrected and ANOVA was repeated on corrected data, in order to verify the modifications of the values. These correction reduced but did not eliminate the differences.

Correlation coefficients were evaluated between the theoretical cheese yield (ThCY), the real yield at 4 (FCY) and 28 h (28CY), the curd dry matter (CDM) and the relationships with the percentage of proteins and milk characteristics. Milk samples were collected every 50 days from 60 buffaloes (326 samples) that were half-sibs (through paternal or maternal lineage ascertained by DNA test), bred in two farms. Positive correlations were found between FCY and 28CY and the enzymatic phase of coagulation and with the milk content in DM, proteins, casein and lipids. Negative correlations were found with lactose and urea milk content. These yields increased when the percentage of DM, proteins and hash of the curd were lower and that of lipids was higher. The 28CY is correlated with ThCY (r = 0.762; P < 0.01) and with the CDM (r = 0.809; P < 0.01). A high 28CY was also characterized by a high 28CY/% proteins ratio and CDM/% proteins ratio. The CDM was higher when the DM of milk and its content in proteins, casein and lipids were higher and when the content in lactose was lower. A correlation (r = 0.501; P < 0.01) was observed between the CDM and the DM percentage of the curd. For the other components the correlations found were the same as for the 28CY. The 28CY/% proteins enhanced at the increasing lipid content of the milk (r = 0.417; P < 0.01) and at decreasing content in urea (r = - 0.317; P < 0.01) and freezing point (r = - 0.123; P < 0.01). The CDM /% proteins was positively influences by fat (r = 0.596; P < 0.01) and lactose (r = - 0.341; P < 0.01). The ThCY/28CY was negatively influenced by “R” and by the pH value and positively correlated with the lipid content of milk.

The aim of this study was to estimate cheese yield by using the chemical- physical parameters of the milk. Analysis were performed on 325 milk samples with 80-219 days in milk interval. Furthermore, buffaloes which showed a ratio between theoretical cheese yield (calculated by Altiero formula) and real cheese yield at 28 hours higher (Group A) or lower (Group B) than 0.983, were compared taking into account 5 hypothetical analytical potentialities of laboratories: 1) Fat percentage; 2) Protein and fat percentages; 3) Protein and fat percentages, pH and SH; 4) Protein and fat percentages, pH, SH, urea, protein percentage corrected per urea, lactose, solids-not-fat (SNF) and SCC; 5) Protein and fat percentages, pH, SH, urea, protein percentage corrected per urea, lactose, SNF, SCC, TAMF, milk DM percentage, ash percentage and casein percentage. Correlation and regression analyses with stepwise method were performed for curd quantity in relation to the physic-chemical ad microbiological milk composition by using SPSS 15.0. As expected, R2 value was such high as the number of variables included in the calculation. A higher R2 value was observed in those samples characterized by a ThCY/28CY ratio < 0.983. ThCY calculated according to Altiero et al (1989), underestimated 28CY of +1.8 g/litre in all samples, whereas a difference between –2.2 (Laboratory 2) and +1.0 (Laboratory 3) g/litre was registered if the actual formula is utilized. According to Altiero formula, 28CY was overestimated of 9.6 g/litre in Group A, whereas it was underestimated of 1.8 g/litre in Group B. According to our study, the estimation of 28CY showed a difference between –9.3 (Laboratory 2) and 9 (Laboratory 1) g/litre in Group A and – 3.5 (Laboratory 1) e 0.0 (Laboratory 5) g/litre.

The aim of this study was to screen for Brucella spp. buffalo embryos produced in- vitro, by using cumulus oocytes complexes (COCs) recovered from ovaries of slaughtered buffaloes naturally infected with Brucella spp. Ovaries were collected from 5 female pluriparous buffaloes slaughtered in a local abattoir. EDTA-blood samples and nasal swabs collected from each animal were used for Brucella spp. DNA detection by real-time PCR. Buffalo ovaries (n = 10) were transported to the laboratory and maintained strictly separated throughout laboratory processing. Recovered COCs were matured, fertilized and cultured in vitro until day 7. Some immature COCs, all uncleaved COCs, all blocked cleaved embryos (2 to 16 cells) and all transferable embryos (tight morulae and blastocysts) were separately analysed by real-time PCR assay. Brucella spp. DNA was detected in both blood and nasal mucus of all subjects, whereas no trace of DNA of Brucella spp. was found on either COCs or embryos. Currently, the infected or seropositive buffaloes have to be slaughtered for sanitary reasons. Interestingly, the results of this preliminary trial suggest a possible utilization of the COCs from the infected subjects of high genetic value to obtain safe embryos.

The aim of this study was to determine if qualitative and quantitative differences exist in energy substrates and ionic components of buffalo cow oviductal fluid during dioestrus, pre-ovulatory and ovulatory phases of the oestrous cycle. Ten multiparous Italian Mediterranean Buffalo (Bubalus Bubalis) cows at 15 days in milk were used. Cannulation of oviduct was performed as previously described by Kavanaugh et al.(1992) for cattle, adapting the technique to the smaller dimension of buffalo reproductive tract. We evaluated daily secretion rates, energy substrates and ions concentration during the three phases. Oviductal fluid secretion rates (ml/24h) and glucose concentration were significantly greater in the ovulatory phase (P

In the last few years, there has been an increasing interest in the in vitro embryo production technologies for faster propagation of superior germplasm in buffalo, due to the low efficiency of multiple ovulation and embryo transfer programs. Early attempts to produce buffalo embryos in vitro have been made by using procedures that were proven effective in cattle. However, the acquisition of more specific information on oocyte and embryo culture requirements in vitro in this species has resulted in an improved efficiency over the years. Although the IVEP efficiency has enhanced, as indicated by competitive embryo yields, pregnancy rate and development to term are still poor. Furthermore, the optimization of embryo cryopreservation methods in this species is critical for the diffusion of ET procedures in the field. The present review intends to describe the state of the art of IVEP in buffalo species, emphasizing the advances achieved and the limitations still to overcome.

The aim of this study was to evaluate the possibility to vitrify in vitro produced (IVP) buffalo and bovine embryos at different stages of development by an advanced version of the “minimal volume approaches”: the Cryotop method. In both experiments, the embryos were vitrified at the tight morula (TM), early blastocyst (eBl), blastocyst (Bl), expanded blastocyst (xBl) and, only for buffalo, at the hatched blastocyst (hBl) stage. After warming, the embryos were cultured in vitro for 24 hours. Stage of development affected the freezability of IVP embryos of both species with the highest embryo survival rates at advanced stages (xBl=76% and hBl=75% for buffalos and xBl=75% for bovine). These results suggest that Cryotop vitrification is an efficient method for buffalo and bovine IVP embryo cryopreservation.

The growing rate of 240 male buffaloes fed ad libitum a diet characterized by 0.9 MFU/Kg dry matter (DM), 14% CP and 38:62 forage:concentrate ratio (Diet A) was evaluated starting from a mean age of 148 (Group 1), 218 (Group 2), 302 (Group 3), 320 (Group 4), 374 (Group 5) and 596 (Group 6) days. Data were compared at the weight of 400 kg and at the age of 550 days, values that were reached by all the subjects. The earlier administration of Diet A increased daily weight gain and reduced DM intake/die and feed conversion index compared to late administration. This trial further confirms the importance of satisfying nutritional requirements during the first months of life in buffalo species, due to the fact that these animals are not able to show a compensative weight gain like bovines.

Here reported are data relative to the chemical composition/characteristics of the milk, of milk samples collected every 50 days from 60 buffaloes (326 samples) that were half-sibs (through paternal or maternal lineage, ascertained by DNA test), bred in two farms (S.A. and M respectively n. = 27 and n = 33). The subjects were divided in relation to the productive level (> vs. < 2601 kg/lactation). The production level did not influence the theoretical yield (ThCY), the yield at 4 h (FCY) and at 28 h (28CY), the curd dry matter (CDM) and the ratios between ThCY and real yields (FCY, 28CY and CDM). Compared to the less productive subjects, despite similar percentage of proteins, the more productive subjects had a higher percentage of casein (3.73 vs. 3.64; P < 0.05), a greater incidence of the latter on total protein (80.52 vs. 79.60; P < 0.05), higher content in lactose (4.88 vs. 4.81; P < 0.01) and urea (42.44 vs. 40.48; P < 0.05) and a lower BCS value (6.79 vs. 7.25; P < 0.05). These differences are not always confirmed in both farms. Regardless of the production level, subjects of farm S.A. had a lower production/lactation (2524 vs. 2817 kg; P < 0.01), higher values of R and k20 (P < 0.01), lower value of a30, a higher SH value and lower contents in urea and solid not fat. They also showed a lower ThCY yield and a lower ratio between ThCY and 28CY when proteins were corrected for the urea content, and a higher ratio between 28CY and the percentage of proteins, both when corrected or not for the urea content. It is difficult to explain the influence of the production level on the casein content whereas the greater lactose content in more productive buffaloes may be accounted for by the greater persistence of lactation curve. The higher urea content found in buffaloes with a greater galactopoietic attitude may be due to a greater DM intake caused by the inadequacy of the diet, hypothesis confirmed by the lower BCS observed in the more productive subjects.

The aim of this study was to verify if the administration of a luteolytic dose of PGF2α at the time of AI is able to anticipate ovulation and, hence, to increase fertility rate. Furthermore, the presence of a “farm effect” was also evaluated. The trial was performed in two commercial farms (A and B) located in Caserta on 584 buffaloes (Farm A: n=389; Farm B: n=195), synchronized by the Ovsynch-TAI Program. In each farm buffaloes were divided in two groups: treated group (n=181 and n=95, respectively in farms A and B) received an intramuscular injection of 0.524 mg cloprostenol (Estrumate®, Schering-Plough Animal health, S.p.A., Milan, Italy) at the time of AI while control group (n=208 and n=100) received an intramuscular injection of physiologic saline (0.9% NaCl). No differences were present with regard to the number of AI/subject, in terms of pregnancy rate and ovulation rate between treated and control groups. It is worth pointing out that the control group in farm A showed a higher (P

The aim of this work was to verify the feasibility of an easy, quick double staining technique for evaluation of frozen-thawed semen to predict the fertilizing capability in vitro of buffalo bulls. In Experiment 1, frozen-thawed semen from 6 bulls was stained with double Trypan blue/ Giemsa and the incidence of acrosome-intact live (AIL), acrosome-intact dead (AID), acrosome-lost live (ALL) and acrosome-lost dead (ALD) sperm was recorded. In Experiment 2, sperm from the same bulls were used to fertilize in vitro matured oocytes. The data obtained confirm that there is a strong “bull effect” in buffalo species, with differences in the percentage of AIL sperm at thawing, in cleavage and blastocyst rates among bulls. Interestingly, it was found that this staining technique can be used for a preliminary screening to select semen to use for IVF, as shown by the correlation existent between the percentages of acrosome-intact viable sperm cells at thawing and the blastocyst yields for 4/6 bulls.

The aim of this work was to evaluate the effects of different warming procedures on the survivability of buffalo in vitro matured oocytes vitrified by the Cryotop (CT) method. In vitro matured oocytes were vitrified in a final solution of 20 % ethylene glycol (EG), 20 % of dimethyl sulfoxide (DMSO) and 0.5 M sucrose. In Group A oocytes (n = 111) were warmed in 1.25 M sucrose for 1 min and then exposed to decreasing concentrations of the sugar (0.625, 0.42 and 0.32 M for 30 sec). In Group B, oocytes (n =122) were warmed into a 0.25 M sucrose solution for 1 min, and then exposed to a 0.15 M sucrose solution for 5 min. Oocytes were rinsed and allocated into the in vitro maturation (IVM) drops for 2 h and then fertilized in vitro. The survival rate was significantly higher in Group A compared to Group B both at 2 h post-warming (92.8 vs 83.6 %; P

The aim of this work was to assess the feasibility of ovum pick-up and in vitro embryo production technology in field conditions in buffalo and crossbreed cattle. This study represents the first trial of in vitro fertilization carried out in North East Argentina. Buffalo (n=11) and bovine (n=3) donors underwent Ovum pick-up twice per week for 2 weeks. The equipment was hand-made, resulting in the impossibility of controlling the aspiration pressure. A higher number of cumulus-oocyte-complexes were recovered in bovine vs buffalo (6.3 vs 3.6 per donor respectively). In bovine the quality of the COCs was also superior (53.9 vs 26.6 % of highest quality COCs in bovine and buffalo respectively). A significantly higher incidence of denuded oocytes was found in buffalo (38.0 vs 11.8 %), indicating a higher sensitivity of buffalo oocytes to mechanical damages. Despite the poor oocyte quality and the suboptimal culture conditions, the latter related to the lack of lab facilities, buffalo embryo cleaved (35.7 %) and developed up to the tight-morula stage (22.5 %). This preliminary trial sets the basis for future studies in Argentina.

In buffalo the overall in vitro embryo production efficiency is lower than cattle, mainly due to the lower cleavage rate (Gasparrini, 2002). In fact in our experience, comparable embryo yields are obtained in the two species only in relation to the cleaved oocytes. It is thought that a major limiting factor is the quality of the frozen semen. It has been demonstrated that freezing results in acrosomal damage, leakage of enzymes, alterations in ionic strength and pH, complete withdrawal of the hydration shell of protein in the solution and loss of motility (Meur et al., 1988). On the other hand we cannot role out that the lower cleavage rate is related to the failure in the accomplishment of oocyte competence after maturation. In fact although nuclear maturation is successfully achieved in our current system.................

In vitro embryo production (IVEP) is largely applied world wide to animal breeding. One of the principal steps of the IVEP is represented by embryo culture (Khurana and Niemann., 2000). In the past, embryos were grown in co-culture systems with other cells such as oviductal epithelial cells, cumulus cells, Buffalo rat liver (BRL) and VERO cells (Duszewska et al., 2000). These cells are able to supply the nutrients for embryo development by their replication and metabolism. Nevertheless, the metabolic activity of these cells is also responsible of an early lowering of pH in the culture medium: that needs to be changed every two days. Furthermore, with this culture system it is impossible to standardize all the procedure: in fact the result is dependent from several variables, as the quality of the cells and their concentration in co-culture. The use of defined culture media is necessary to acquire a better comprehension of metabolism and biochemical requirements for IVEP........

Aim of this study was to evaluate the mineral milk content and its relationship with the cheese yield and the rennet coagulation properties. Ca and P content, total and soluble, was determined on 70 milk samples along with the physic-chemical composition, cheese yield and coagulation parameters. Total Ca and P contents were 170,57±14,41mg·dl-1 and 145,34±26,87 mg·dl-1,with a Ca/P ratio of about 1.2. Fresh cheese yield was on average 261.7 ± 25.4 gr·l-1 of milk and was influenced by both milk quality and Ca and P contents (R2=0.82). The average rennet coagulation parameters had the following values: R = 14,20±3,82; K20= 1,73±0,97 and A30= 46,01±8,81. R values was influenced positively (R2=0.68) by milk pH, protein and fat content and negatively by the Ca/P ratio while shorter K20 value were linked to low micellar Ca and higher soluble P (R2=0.46). The A30 was negatively influenced by milk pH, fat/protein ratio and positively by soluble Ca and P content and micellar P % (R2=0.50).

Aim of this study was to verify the relation among the mediators and indicators of nutritional status like insulin, glucagon, urea, cholesterol, triglycerides and total proteins in growing buffalo heifers, fed diets with different energy density. 12 Murrah heifers were randomly allocated into two dietary treatments (High, Group H; Low, Group L) that differed in energetic levels (Group H: 5.8 UFL/d; Group L: 3.6 UFL/d). Every 30 days, for a total of five times, blood samples were collected at 08.00 h, before feeding, from the jugular vein in vacutainer tubes and analysed to determine metabolic profile. Data on haematic constants were analysed by ANOVA for repeated measures with treatment as the main factor. Low energy availability and low NSC reduced the glucose and insulin and increased glucagone and urea blood levels. The increase of NSC in the diet of group H during the experiment may caused a reduction of the fibre digestibility after the period of adaptation of the rumen microflora and, as a paradox effect, suffered for an energetic lack with a subsequent activation of lipolysis and mobilization of their body reserves. Liver and muscular synthesis increase in group with a high energy availability.

Although great importance has been recently given to the in vitro embryo production (IVEP) techniques in buffalo species, the overall efficiency is still low because many physiological aspects of oocytes/embryos have not been properly investigated. It has been speculated that buffalo oocytes/embryos, due to their high lipid content (Boni et al., 1992), are particularly sensitive to the increased oxidative stress, that occurs under in vitro conditions (Gasparrini et al., 2003).

In buffaloes, an optimal BCS at calving time improves reproductive efficiency (Baruselli et al., 2001) therefore shortening the calving/conception period and lowering the number of services/conception. In buffalo cows in negative energy balance a delayed ovulation and a reduced percentage of large follicles were found (Campanile et al., 2001). In buffaloes naturally mated protein degradability in the rumen did not influence reproductive activity (Campanile et al., 2003). It is possible that, independently of the Blood Area (BU), a lower diffusion of ammonia occurs in the uterus, reducing the detrimental effect on reproductive efficiency. The aim in the present study was to determine the influence of BCS, urea e ammonia blood levels on conception rate in Italian Mediterranean buffaloes synchronised and mated by AI in mid-winter which coincided with transition to the seasonal nadir in reproductive activity.

The aim of the present study was to evaluate the effect of melatonin supplementation during in vitro maturation on fertilization and embryo development in cattle. Bovine cumulus-oocyte-complexes (COC), recovered from abattoir ovaries, were matured in vitro in the absence (control) and in the presence of 10 μM, 100 μM and 1 mM of melatonin. Matured oocytes were fertilized in vitro with frozen-thawed sperm and cultured up to the blastocyst stage. The results of this work demonstrated that melatonin enrichment of the in vitro maturation (IVM) medium does not affect both cleavage (71.0, 72.8, 72.5 and 72.7 % in the control group and in the groups supplemented with 10 μM, 100 μM and 1 mM of melatonin respectively) and blastocyst rates (41.3, 33.8, 39.4 and 38.3 % respectively) in cattle.