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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.

It was previously demonstrated that the kinetics of early cleavage could be used to discriminate between bovine bulls with high and low field fertility (Ward F et al.2001 Mol. Reprod. Dev. 60, 47–55). Marked differences exist in the kinetics of sperm penetration between bulls, and this may be a useful predictor of field fertility in cattle (Ward F et al.2002 Theriogenology 57, 2105–2117). It is well known that the ability to fertilize oocytes in vitroand to sustain embryo development varies significantly among buffalo bulls. Therefore, the aim of this work was to evaluate whether the speed of oocyte penetration after IVF was correlated with the blastocyst rates obtainable with different bulls in buffalo species. In Experiment 1, in vitro-matured buffalo oocytes were co-incubated with MitoTracker-labeled spermatozoa (Ward F et al.2002 Theriogenology 57, 2105–2117) from 6 different bulls, over 2 replicates. Oocytes were subsequently fixed every 3 h (up to 18 h) postinsemination (pi). At each time point, oocytes were denuded, dezoned, fixed in ethanol overnight, and stained with 4′,6-diamidino-2-phenylindole for nuclei examination under a fluorescence microscope. In Experiment 2, in vitro-matured oocytes were fertilized with sperm from the same 6 bulls and were cultured to the blastocyst stage, over 4 replicates. Bulls were tested, collectively, on each batch of ovaries in both experiments. Differences in the percentages of monospermic penetration among bulls were analyzed by chi-square test. Correlation and multiple regression analyses were also carried out between the speed of penetration and blastocyst yields. Marked differences in the kinetics of sperm penetration were found among buffalo bulls, as shown in Table 1. Interestingly, a correlation was found between the blastocyst rate and the percentage of oocytes penetrated at 6 h (r = 0.71; P< 0.01), at 9 h (r = 0.65; P< 0.05), at 12 h (r = 0.77; P< 0.01), and at 18 h pi (r = 0.59; P< 0.05). Regression analysis showed that the optimal time of penetration for predicting the blastocyst rate was 12 h pi (R2= 0.6). In conclusion, the kinetics of sperm penetration may be a useful marker to predict the in vitro-fertilizing ability of buffalo bulls. The great variability in the speed of oocyte penetration suggests inserting this assessment in the preliminary screening of bulls before their utilization in IVF programs. This may be helpful in selecting high-fertility bulls and identifying the optimal gamete co-incubation times for each bull used.Table 1.Percentage of oocytes penetrated at each time point (hpi, h postinsemination) by different bulls1 [ABSTRACT FROM AUTHOR]

It was previously reported that osteopontin (OPN), an acidic single-chain phosphorylated glycoprotein found in the oviductal fluid in cattle (Gabler C et al.2003 Reproduction 126, 721–729), is able to facilitate fertilization in this species (Gasparrini B et al.2008 Reprod. Fertil. Dev. 20(Suppl. I), 180 abst). The present study aimed to investigate whether the addition of OPN to the fertilization medium would affect both cleavage and postfertilization embryo development in the buffalo. To assess the influence of OPN on cleavage and blastocyst rates, in vitro-matured oocytes were fertilized in modified Tyrode''s albumin lactate pyruvate medium (Lu KH et al.1987 Vet. Rec. 121, 259–260) supplemented with penicillamine, hypotaurine, and heparin, in the presence of 0.0 (n= 258), 0.1 (n= 263), 1 (n= 261), and 10 μg mL-1(n= 264) of OPN. In vitrofertilization was carried out with frozen–thawed spermatozoa from a bull already tested for IVF. After 20 to 22 h of co-incubation at 38.5°C and 5% CO2in air, putative zygotes were gently pipetted to remove cumulus cells, washed, and transferred, 10 per droplet, into 20 μL of SOF medium including essential and nonessential amino acids and BSA (Tervit HR et al.1972 J. Reprod. Fertil. 30(3), 493–497), in a controlled gas atmosphere consisting of 5% CO2, 7% O2, and 88% N2, in humidified air, at 38.5°C. The culture medium was changed on Day 5 (Day 0 = day of insemination), when cleavage rate was assessed and embryos were moved into fresh medium for an additional 2 days. On Day 7, development rates into blastocysts of superior quality were recorded. Differences in the percentages of both cleavage and blastocyst rates among groups were analyzed by chi-square test. Significantly higher cleavage rates (59.3, 70.3, 71.6, and 42.4%, respectively, in the control group and in the groups with 0.1, 1, and 10 μg mL-1of OPN; P< 0.01) were observed in the groups with 0.1 and 1 μg mL-1of OPN compared with the other groups. Likewise, higher blastocyst rate percentages (17.4, 27.4, 29.9, and 9.5%, respectively, in the control group and in the groups with 0.1, 1, and 10 μg mL-1of OPN; P< 0.01) were observed in the groups with 0.1 and 1 μg mL-1of OPN compared with the other groups. In conclusion, these results showed that addition of low concentrations of OPN in the fertilization medium improved both cleavage and postfertilization embryo development in the buffalo, whereas the higher concentration resulted in impaired late-stage embryo development. [ABSTRACT FROM AUTHOR]

The overall in vitro embryo production efficiency in buffalo is hampered by the poor fertilization rate. It is known that the quality of the frozen semen may affect fertilization efficiency. However, it is not possible to rule out that the process of capacitation, required by spermatozoa to acquire the fertilizing ability, is impaired in the in vitro fertilization (IVF) system. Although several agents have been proven to induce sperm capacitation in vitro, heparin treatment is still the most efficient method in most of the domestic species. There is evidence that capacitation is part of an oxidative process and that nitric oxide (NO) acts as a capacitation inducer in human (Herrero et al. 1999 Biol. Reprod. 61, 575–581) and bovine (Rodriguez et al. 2005 Anim. Reprod. Sci. 85, 231–242) spermatozoa. The aim of the present study was to evaluate whether sodium nitroprusside (SNP), a well-known generator of NO in vitro, improves buffalo sperm capacitation in vitro. Frozen–thawed sperm from a bull previously tested for IVF were treated by swim-up in order to select only the motile population. Spermatozoa were incubated in the presence of 0.01 mM heparin (control group) for 1 h (n = 266), 2 h (n = 270), and 3 h (n = 306), and in the presence of 10 M SNP for 1 h (n = 302), 2 h (n = 286), and 3 h (n = 260). The concentration of SNP was chosen on the basis of a preliminary dose-response trial (0.1 M, 1 M, and 10 M). Following incubation with these agents, sperm were exposed for 15 min to 60 g mL-1 of lysophosphatidylcholine, an agent known to induce acrosome reaction only on capacitated spermatozoa. Trypan blue was used first to differentiate live from dead spermatozoa and the dried smears were then fixed in 37% formaldehyde and stained with Giemsa for acrosome evaluation by microscopic examination. The proportion of acrosome-reacted spermatozoa in each group was used to assess the efficiency of capacitation under different incubation conditions. Differences between groups were analyzed by chi-squared test. No dead spermatozoa were found in all groups. Following 1-h sperm treatment with either heparin or SNP, the proportion of acrosome-reacted spermatozoa was similar (35.3% vs. 28.5%, respectively). However, extending the incubation time to 2 h, SNP significantly (P < 0.01) increased the incidence of acrosome reaction compared to heparin (60.1% vs. 44.1%, respectively). Analogously, when the sperm treatment was prolonged to 3 h, SNP gave a significantly (P < 0.01) higher percentage of acrosome reaction compared to the control (68.8% vs. 36.6%, respectively). In conclusion, sperm treatment with SNP for either 2 or 3 h significant improved the efficiency of buffalo sperm capacitation in vitro compared with heparin, that is, the capacitating agent currently used in the IVF system. The promoting effect of SNP indirectly indicates that NO acts as a capacitation inducer in buffalo spermatozoa. Finally, these results suggest the need to evaluate the effect of SNP on the fertilizing capability of buffalo spermatozoa in vitro. [ABSTRACT FROM AUTHOR]

The aim of this study was to compare the efficiency of different combinations of cryoprotectants for vitrification of IVP buffalo (Bubalus bubalis) embryos by the cryotop method (Kuwayama et al. 2005 RBM Online 11, 300–308). In group A, we evaluated the vitrification and warming solutions previously used to vitrify buffalo embryos in French straws (Gasparrini et al. 2001 Theriogenology 55, 307). Embryos were equilibrated in 1.4 M glycerol for 5 min and in 1.4 M glycerol and 3.6 M ethylene glycol (EG) for an additional 5 min. After being transferred into 3.4 M glycerol and 4.6 M EG for 25 s, individual embryos were picked up in an extremely small volume (<0.1 L) of vitrification solution and placed on the top of a very fine polypropylene strip (0.4 mm wide 20 mm long 0.1 mm thick) attached to a hard plastic handle, kindly provided by M. Kuwayama. Each embryo was placed onto the thin strip of the Cryotop and immediately submerged into liquid nitrogen. For warming, the strip of the Cryotop was immersed directly into a 0.5 M sucrose solution; embryos were retrieved and transferred into 0.25 M sucrose for 5 min before culture in SOF medium. In group B, we examined the vitrification and warming solutions previously used for OPS vitrification of buffalo embryos (De Rosa et al. 2006 Reprod. Fertil. Dev. 18, 153). Embryos were equilibrated in 7.5% EG + 7.5% dimethyl sulfoxide (DMSO) for 3 min before transfer into 16.5% EG + 16.5% DMSO + 0.5 M sucrose. After 25 s, they were placed on the cryotop, as previously described, and submerged into liquid nitrogen. For warming, embryos were recovered into a 0.25 M sucrose solution for 1 min, transferred into 0.15 M sucrose for 5 min, and cultured in SOF. IVP buffalo embryos of excellent quality that, by Day 7 of culture (Day 0 = in vitro fertilization), had reached the blastocyst stage (n = 44 and 53 for groups A and B, respectively), over 6 replicates, were vitrified. Embryo survival rate was determined as the percentage of vitrified-warmed embryos undergoing further development during a 24-h in vitro culture period. Differences between methods were analyzed by chi-square test. A significantly higher embryo survival rate was recorded in Group B compared to Group A (67.9 vs. 43.2% respectively; P < 0.05). In conclusion, it was demonstrated that cryotop vitrification, with the combination of cryoprotectants used in group B, is a valid tool to cryopreserve IVP buffalo blastocysts. [ABSTRACT FROM AUTHOR]

Despite the increasing interest, buffalo oocyte cryopreservation is still inefficient, especially in terms of blastocyst development after IVF. The aim of this work was to evaluate chromatin and spindle organization of buffalo in vitro-matured oocytes after vitrification/warming by cryotop and after their simple exposure to cryoprotectants (CP). An overall amount of 251 COC was selected and matured in vitro. In the vitrification group, COC were first exposed to 10% ethylene glycol (EG) + 10% DMSO for 3 min, and then to 20% EG + 20% of DMSO and 0.5 msucrose, loaded on cryotops, and plunged into liquid nitrogen within 25 s. Oocytes were warmed into a 1.25 msucrose solution for 1 min and then to decreasing concentrations of sucrose (0.625 m, 0.42 m, and 0.31 m) for 30s each. In order to test CP toxicity, COC were simply exposed to the vitrification and warming solutions. Two hours after warming, oocytes were fixed and immunostained for microtubules using a method previously described (Messinger SM and Albertini DF 1991 J. Cell Sci. 100, 289–298), stained for nuclei with Hoechst, and examined by fluorescence microscopy. Fresh in vitro-matured oocytes were fixed and stained as controls. Data were analyzed by chi-square test; results are shown in Table 1. The percentages of MII oocytes in the control and vitrification groups were greater than in the toxicity group, in which a greater percentage of telophase II stage oocytes were found compared with both the control and vitrification groups, indicating occurrence of activation. Of the MII oocytes, both exposure to CP and vitrification procedures gave greater percentages of oocytes with abnormal spindle and abnormal chromatin configuration compared with the control. An unexpected datum was the evidence of a significant percentage of spontaneously activated oocytes in the toxicity group. We speculate that the lack of activation in the vitrification group may be related to the slowing down of metabolic activity subsequent to thermal shock, and hence, that activation after vitrification may occur later than 2 h post-warming. In conclusion, the simple exposure to CP causes activation of the COC and damage to the cytoskeleton similar to that induced by the whole vitrification protocol. The damages to the meiotic spindle and DNA fragmentation may lead to aneuploidy incompatible with subsequent embryo development and account for the poor embryo development currently recorded in buffalo.Table 1.Chromatin and spindle organization in oocytes vitrified and exposed to cryoprotectants [ABSTRACT FROM AUTHOR]

The establishment of porcine pluripotent ES cell lines would be an exciting and novel tool for animal biotechnology, such as cloning and transgenesis. Furthermore, it would represent a useful model for biomedical research, cell therapy, xenotransplantation as well as developmental biology research. However, in spite of several studies, no conclusive results have been obtained and a number of technical questions are still to be answered in order to derive genuine ES cells in the pig. Here we report the results obtained in our laboratory aimed at comparing IVF v. parthenogenetic embryos as a source for the establishment of putative ES cells. Oocytes were divided in two groups and subjected to IVF or parthenogenetically activated with ionomicyn and 6-DMAP. They were cultured in NCSU for 7 days and then subjected to immuno-surgery. Inner cell mass were plated onto inactivated STO feeder cells and outgrowth formation was monitored. Cells were passaged to a new STO monolayer every 7 days. Assessment of pluripotency markers was carried out both by RT-PCR and immunocytochemical analysis at every passage for up to 22 passages. Telomerase activity was measured every 5 passages. The results indicate that parthenogenetic embryos, although less resilient than IVF embryos to immuno-surgery, have a significantly greater ability to generate outgrowths and stable cell lines. Moreover, 77% of the 39 parthenogenetic lines derived v. only 33% of the IVF ones expressed pluripotency markers and displayed high telomerase activity. Altogether our findings are consistent with data obtained in the human where the efficiency to derive hES cell lines from parthenogenetic blastocysts appears greater as compared with regular blastocysts from IVF embryos (Cheng L 2008 Cell Research 18, 215–217).Table 1.Supported by: Prin 2005, 2006. [ABSTRACT FROM AUTHOR]

Osteopontin (OPN) is an acidic single-chain phosphorylated glycoprotein found both in the oviduct fluid (ODF) and oviductal epithelium in cattle, which is believed to facilitate fertilization. It was recently reported that addition of a rabbit polyclonal immunoglobulin G antibody against purified bovine milk OPN with sperm oocytes, bovine oocytes, or both decreased (P < 0.05) fertilization compared with the in vitro-fertilized control (Goncalves et al. 2007 Theriogenology 67, 468–74). The aim of the present work was to determine the effect of in vitro addition of OPN to the fertilization medium on both cleavage and postfertilization embryo development in cattle. In the first experiment, in vitro-matured oocytes were fertilized in modified TALP medium in the presence of 0.0, 0.1, 1.0, or 10 µg mL–1 of OPN. In a second experiment, matured oocytes were in vitro-fertilized in modified TALP medium in the presence of 0.0, 10, 20, or 40 µg mL–1 of OPN. In vitro fertilization was carried out with frozen–thawed spermatozoa from a bull previously tested for IVF. After 20 to 22 h of coincubation at 39C, 5% in CO2 in air, presumptive zygotes were vortexed to remove cumulus cells, washed, and transferred, 30 to 50 per well, into 400 µL of SOF modified medium. Zygotes were incubated in a humidified mixture of 5% CO2, 7% O2, and 88% N2 in air at a temperature of 39C. On Day 7 of development (Day 0 = day of insemination), cleavage and development rates into transferable embryos (TE)–tight morulae (TM) and blastocysts (Bl) of superior quality were recorded. Differences in the percentages of both cleavage and blastocyst rates among groups were analyzed by a chi-square test. In experiment 1, numerically higher percentages of TM–Bl (29.5, 29.5, 30.5, and 37.5%, respectively, in the control group and in the groups with 0.1, 1, and 10 µg mL–1 of OPN; P = 0.25) and Bl (28.6, 27.5, 29.1, and 36.7, respectively, in the control group and in the groups with 0.1, 1, and 10 µg mL–1 of OPN; P = 0.24) were observed with 10 µg mL–1 of OPN. In experiment 2, significantly more cleavage (80.0 v. 71.3%; P < 0.05) and higher percentages of TM–Bl (44.6 v. 34.5%; P < 0.05) and Bl (39.2 v. 30.6%; P = 0.06) were observed with 10 µg mL–1 of OPN v. the control. Combined analysis from both experiments showed an overall effect of 10 µg mL–1 of OPN v. the control in the percentages of TM–Bl and Bl (respectively, 41.1 v. 33.3% and 37.7 v. 30.5%; P < 0.05). These results indicate that it is possible to improve the efficiency of bovine in vitro embryo production by adding the oviductal protein OPN. [ABSTRACT FROM AUTHOR]

Although removal of cumulus cells improves the efficiency of vitrification of buffalo (Bubalus bubalus) in vitro-matured (IVM) oocytes (Gasparrini et al. 2007 Anim. Reprod. Sci. 98, 335–342), the lack of cells impairs the fertilization process. Therefore, the aim of the present work was to evaluate the influence of a somatic support during in vitro fertilization (IVF) of buffalo vitrified denuded matured oocytes. Since IVF on a cumulus cells monolayer was inefficient, we verified the effects of co-culture with cumulus-enclosed oocytes (COCs). IVM buffalo oocytes (n = 316) were vitrified by the Cryotop method (Kuwayama and Kato 2000, J. Assist. Reprod. Genet. 17, 477 abst) that was recently proven suitable for buffalo oocyte cryopreservation (Attanasio et al. 2006 Reprod. Domest. Anim. 41, 302–310). Denuded buffalo oocytes were equilibrated in 10% ethylene glycol (EG) and 10% dimethyl sulfoxide (DMSO) for 3 min, transferred into 20% EG and 20% of DMSO in TCM199 with 20% fetal calf serum (FCS) + 0.5 m sucrose, loaded on Cryotops, and plunged into liquid nitrogen within 25 s. For warming, oocytes were exposed for 1 min to 1.2 m sucrose and then to decreasing concentrations of the sugar (0.6, 0.4, 0.3 m for 30 s) in TCM199 + 20% FCS. Oocytes were rinsed and allocated to IVM drops for 1.5 h. Survival rate was evaluated at this point and the oocytes that had survived (292/316 = 92.4%) were split into 2 fertilization groups: (A) approximately 5 buffalo oocytes per 50-µL drop of IVF medium, and (B) approximately 3 buffalo oocytes + 3 bovine fresh COCs per 50-µL drop of IVF medium. Since buffalo COCs easily lose their cells following IVF, for better identification we used bovine COCs that have a brighter and more compact cumulus mass. In vitro fertilization and culture were carried out as previously described (Gasparrini et al. 2007). As control, buffalo oocytes (n = 104) were in vitro-matured, fertilized, and cultured up to the blastocyst stage. On Day 1, survival rate was evaluated in the two vitrification groups; cleavage and blastocyst rates were recorded on Days 5 and 7, respectively, in all groups. The experiment was repeated 4 times. Differences in the percentages of survival, cleavage, and blastocyst formation among treatments were analyzed by chi-square test. Within vitrification groups, despite similar survival rates on Day 1 (90.6% v. 93.3%, respectively, in Groups A and B), cleavage rate was significantly improved in Group B compared to Group A (59.2% v. 45.4%, respectively; P < 0.01). Interestingly, the cleavage rate in Group B was not significantly different from that recorded in the control group (71.0%). Although blastocysts were produced in both vitrification groups (3.6% v. 4.1%, respectively, in Groups A and B), the yield was significantly lower than that of the control group (29.0%, P < 0.01). In conclusion, co-culture with bovine COC during fertilization improves the capability of buffalo denuded vitrified oocytes to cleave. [ABSTRACT FROM AUTHOR]

The competitiveness of buffalo breeding will depend on the utilization of reproductive biotechnologies that allows acceleration of genetic progress. A major factor hampering the efficiency of both artificial insemination and in vitro embryo production programs in this species is male hypofertility. Reports for several species suggest that seminal plasma contains factors that influence male fertility. Osteopontin is a glycoprotein found in several biological fluids including seminal plasma, and its presence is associated with spermatozoa concentration. In cattle, expression of osteopontin was highly correlated with bull fertility, and it was proposed to be a marker to predict male fertility (Cancel et al. 1999 Biol. Reprod. 60, 454–460). No data are available about the presence or activity of osteopontin in water buffalo. The aim of this preliminary study was to determine if osteopontin is present in buffalo semen and to evaluate whether freezing procedures cause the loss of osteopontin from spermatozoa. Semen was collected in authorized semen collection centers from 6 buffalo bulls by using an artificial vagina. A collection of bovine semen was used as a positive control. An aliquot from each sample was frozen using standard procedures for semen storage. Each ejaculate was centrifuged at 600g for 10 min at room temperature, and the supernatant was recovered and centrifuged at 10 000g for 1 h at 4C. The total protein concentration in seminal plasma and spermatozoa was determined by the Bradford method, using ovoalbumin as the standard. Proteins (50 g) were separated by electrophoresis and analyzed by western blotting (Cancel et al. 1999). Polyclonal antibodies against bovine milk osteopontin were prepared as previously described (Cancel et al. 1997 Biol. Reprod. 57, 1293–1301). The intensities of bands indicated by western blot were quantified by densitometer. Osteopontin was detected in all samples of buffalo semen. Most of the osteopontin detected was in the seminal plasma. Relative amounts of osteopontin detected in spermatozoa were 50% or less of that detected in seminal plasma; furthermore, the protein was not found in sperm from all bulls. These results suggest that most osteopontin is produced by the ampullae and seminal vesicles, similar to what was reported for cattle (Cancel et al. 1999). Semen frozen by standard procedures showed a reduction in amount of osteopontin by up to 50%. These studies suggest that the fertility-associated protein osteopontin may be useful as a sensitive tool to evaluate whether sperm storage procedures are detrimental and result in excess loss of osteopontin from sperm. In conclusion, the results have demonstrated that osteopontin is present in buffalo seminal plasma and sperm. Further studies will examine whether the expression of osteopontin is correlated with the fertility of buffalo bulls, as has been demonstrated in bovine bulls. [ABSTRACT FROM AUTHOR]

In high-producing dairy cows periparturient negative energy balance (NEB) triggers body fat mobilization, resulting in elevated blood levels of non-esterified fatty acids (NEFAs). Blood is dominated by the saturated fatty acids (SFA) palmitic (C16:0) and stearic acid (C18:0), which are associated with a negative effect on oocyte developmental competence. In contrast, the monounsaturated fatty acid (MUFA) oleic acid is harmless and is able to counteract the negative effect of saturated NEFAs on in vitro maturing oocytes. Since preantral follicles lack oleic acid-rich follicular fluid, we hypothesized that preantral follicles and oocytes may benefit from oleic acid-rich fat supplementation during NEB. Eight-month pregnant Holstein Friesian heifers were randomly divided in two groups to receive a standard, palmitic acid-rich (CTR, n = 5), or rumen-protected oleic acid-rich (UNSAT, n = 6), periparturient fat supplementation until 4 weeks post-calving. NEFA, β-Hydroxybutyric acid and haptoglobin profiles in blood were monitored, and cumulus-oocyte-complexes (COCs) were via transvaginal ovum pick-up (OPU) collected at 8, 12, and 16 weeks postpartum for in vitro maturation, fertilization (day 0), and culture until day 8. Oleic acid supplementation increased C18:1 and reduced C16:0 levels in blood in comparison to CTR, during the peripartum period. Interestingly, the UNSAT group exhibited a 1.6-times higher oocyte yield in comparison to the CTR, but no difference in oocyte developmental competence between the groups. These findings suggest that peripartum oleic acid supplementation supports follicles and oocytes during NEB. Potential long-term benefits of oleic acid on fertility in dairy cows, in a higher number of animals, warrant further investigation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.)

This work aimed to evaluate whether supplementing the freezing extender with olive fruit extract (OFE) would improve the antioxidant defense of buffalo sperm, resulting in improved post-thaw semen quality. Ejaculates (two per 16 Murrah buffalo bulls) were split into four aliquots that were diluted in an extender supplemented with different doses of OFE (0, D50, D100, and D150, based on µM concentrations of hydroxytyrosol, the most represented polyphenol) and frozen according to standard procedures. At thawing, sperm motility, kinetics, viability, acrosome integrity, and membrane functionality were evaluated. Based on the dose-response results, biological antioxidant potential (BAP) and reactive oxygen metabolites (ROMs) were assessed after thawing in D50 and control groups. The pre-freezing supplementation of the extender with D50 OFE showed higher (P < 0.05) total and progressive sperm motility, as well as straight-line velocity compared to the control. Treatment with D50 OFE of buffalo semen also improved (P < 0.01) post-thaw sperm viability, membrane functionality, and acrosome integrity compared to the control. The enrichment of the extender with D50 OFE increased (P < 0.01) the post-thaw BAP and reduced (P < 0.05) the ROMs levels. The highest concentration tested (D150 OFE) negatively affected (P < 0.05) total and progressive motility, and the percentage of sperm with functional membranes and intact acrosomes, compared to the control. In conclusion, low doses of OFE added to the extender significantly improved post-thawing buffalo semen quality by protecting the spermatozoa from cryopreservation-induced oxidative stress. Further studies should investigate its effectiveness on in vivo and in vitro fertility, for potential commercial applications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 The Authors. Published by Elsevier B.V. All rights reserved.)

Freezing-thawing procedures and semen manipulation for in vitro fertilization induce oxidative stress, which in turn leads to impaired sperm quality. The aim of this study was to evaluate whether incubation of frozen-thawed buffalo semen with olive fruit extracts (OFE), known to contain a high concentration of phenolic antioxidants, would improve semen quality by reducing oxidative stress. Frozen sperm (4 ejaculates/4 bulls/3 replicates) were thawed and diluted to 30 × 10 6 /mL in IVF medium with 0, 72, 143, and 214 μL/mL of OFE, corresponding to 0 (D0-control), 50 (D50), 100 (D100), and 150 (D150) μM hydroxytyrosol. Sperm viability, acrosome integrity, membrane functionality, motility, and sperm kinetics were evaluated immediately after thawing (T0) and after 1 (T1) and 2 h (T2) of incubation at 38.7 °C. Based on the results, sperm biological antioxidant potential (BAP) and ROS levels (ROMs) were assessed in D0 and D100 groups at T1 and T2. To assess the effect of OFE on fertilizing ability, heterologous penetration rates were also evaluated, using bovine abattoir-derived oocytes. The treatment with OFE at all concentrations tested increased (P < 0.05) the percentage of acrosome intact spermatozoa compared to the D0-control at T1, but the effect was more evident (P < 0.01) with D100 (54.5 ± 3.0, 60.5 ± 1.5, 65.2 ± 3.3, and 62.5 ± 1.7, with D0, D50, D100, and D150 OFE, respectively). Total motility, progressive motility, rapid velocity, and progressive velocity decreased (P < 0.05) at T2 only in the D0-control group. The percentage of rapidly progressive sperm and the progressive motility tended to increase (P < 0.10) at T1 and T2, respectively, in D100 compared to D0 (24.7 ± 4.1 vs 16.4 ± 1.6 and 22.8 ± 2.7 vs 17.0 ± 1.2, respectively). The treatment with D100 OFE of frozen-thawed sperm increased (P < 0.05) some kinetic parameters (VAP and WOB). Spermatozoa incubated with D100 OFE exhibited higher (P < 0.01) total and normospermic oocyte penetration rates compared to D0 (86.5 ± 1.4 vs 78.5 ± 0.7, and 70.6 ± 1.5 vs 63.8 ± 1.1, respectively). Additionally, D100 OFE increased sperm BAP concentrations at both T1 and T2, while ROS levels were unaffected. These results suggest that incubating frozen-thawed buffalo semen with OFE is an effective strategy for preserving semen quality and in vitro fertilization ability by enhancing sperm antioxidant capacity., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Defining the optimal therapy for endometritis remains a significant challenge for clinicians. Given the public health threat posed by antibiotic resistance and the inconclusiveness of traditional therapies, regenerative medicine has been proposed as an alternative. The objective of this study was to conduct a comprehensive systematic review and meta-analysis, to investigate the efficacy of regenerative medicine products in the treatment of both post-breeding persistent and chronic degenerative endometritis (PBIE/CDE) in mares, following the PRISMA guidelines. This research could be a comprehensive scientific reference for determining appropriate treatments and clinical strategies. All studies exploring the use of regenerative medicine therapies (i.e., plasma products, autologous conditioned serum, mesenchymal stem cells MSCs, and MSC derivatives) in the treatment of PBIE/CDE were included, regardless of the specific protocol used, the evaluated outcomes, or the diagnostic method employed. Two authors independently gathered data and evaluated the risk of bias for each study. Treatment effects were assessed using risk ratios for dichotomous data, accompanied by 95 % confidence intervals. Data were aggregated utilizing the fixed-effects model. The quality of evidence for each outcome was evaluated using GRADE criteria. Eighteen studies were included in the systematic review, while fifteen trials were included in the meta-analysis. A sub-meta-analysis was conducted separately on platelet-derived products, as well as on MSCs and their derivatives. The results demonstrated an overall positive effect of regenerative therapies in treating PBIE/CDE, particularly those involving MSCs and their derivatives. The positive outcomes include an anti-inflammatory effect, characterized by reduced intrauterine fluid accumulation, neutrophils, and cytokine concentrations. Additionally, improvements in pregnancy, foaling, and embryo recovery rates have been observed in some cases. Despite the limited number of randomized controlled studies and the high variability among protocols, including the timing of treatment, type, and volume of products used, the use of regenerative products, especially MSCs and their derivatives, has promising results in terms of both efficacy and safety for treating PBIE/CDE in mares., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)