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The aims of the investigation were to establish for the first time (i) clinical efficacy and (ii) pharmacokinetic profile of meloxicam intravenously (IV) administered in male Mediterranean buffalo calves after surgical orchiectomy. The study was performed on 10 healthy buffalo calves, between 4 and 5 months old and between 127 and 135 kg of body weight (b.w.). An IV injection of 0.5 mg/kg b.w. of meloxicam was administered in six calves (treated group, TG) immediately after surgery; the other four animals were used as untreated control group (CG). The clinical efficacy of meloxicam was evaluated pre- and post-surgery by monitoring respiratory rate (RR), heart rate (HR), rectal temperature (T°C), serum cortisol levels (SCL) and pain score (PS). Significant inter-groups differences were detected at sampling times (T): 4 hour (h) for RR (P

The buffalo species represents the mainstay of the rural economy of small farmers in many developing countries, and is also an important source of protein. Because of its area of origin, it shows some peculiar characteristics regarding protein requirements and nitrogen balance. Therefore, this review aims at clarifying some aspects of protein digestibility and, consequently, manure management in buffalo cows.

The peculiar reproductive characteristics of the species limit the utilization of artificial insemination (AI) in buffalo. For this reason, in the last years a great attention has been focused on estrus synchronization methods based on fixed timed artificial insemination (FTAI). Among these, the Ovsynch-TAI program has been successfully applied in buffalo. The aim of this study was to assess the response to the first GnRH, prostaglandin and second GnRH in buffaloes synchronized by Ovsynch and subsequent pregnancy outcome. The trial was carried out on 59 buffaloes (138.7±86 days in milk) bred in a commercial farm in the South of Italy. Ten days before the start of the study all the animals underwent clinical and ultrasound examination, to confirm the absence of any gross abnormalities of the genital tract. Synchronization of the estrous cycle was performed by Ovsynch-TAI program which involved the injection of a GnRH agonist on Day 0 (12 μg of buserelin acetate), prostaglandin on Day 7 (5 mg of Dinoprost), and a GnRH agonist again on Day 9 (12 μg). The AI was carried out 16 h after the second GnRH injection, using frozen-thawed semen from bulls of proven fertility. The ultrasound examinations of the ovaries were performed by using a portable Sonoace Pico equipped with a 10 MHz linear transducer adapted for transrectal examination in large domestic animals on Day 0, 1, 7, 9, 10 and 11. All visible antral follicles were recorded and classified into three categories, according to their size: small (diameter < 0.5 cm), medium (diameter between 0.5 and 1 cm) and large (diameter > 1cm). The dimensions of the dominant follicle and the corpus luteum (CL) were further registered. In order to evaluate the characteristics of the CLs, the colour-doppler mode was activated in order to display signals for blood flow in vessels. Pregnancy diagnosis was carried out on 35 days post-AI and the statistical analysis was performed by ANOVA and chi-square test. It was observed that 37 buffaloes (62.7%) ovulated after the first GnRH treatment and showed a functional corpus luteum on day 7. Interestingly, animals that ovulated after the first GnRH injection showed a lower (P=0.08) CL area (22.1±10.1 vs. 28.6±10.3 mm, respectively) and a larger follicle (10.5±4.3 vs. 8.5±3.0, respectively) on day 0. A total of 54 buffaloes (91.5%) showed a functional CL on day 7, although luteolysis occurred only in 50 subjects (84.7%). All the buffaloes (37/37) that ovulated after the first GnRH injection underwent luteolysis. On the contrary, CLs responsive to prostaglandins were recorded only in 59% (n=13) of the animals that did not ovulate after the first GnRH (P

The aim of this study was to evaluate water balance and nitrogen excretion in Mediterranean buffaloes. Sixteen subjects were divided into 4 groups (n=4/group), according to their days in milk (DIM): Group L50 (DIM=50±8); Group L125 (DIM=125±55); Group L225 (DIM=225±26); Group NL (non-lactating). The study comprised a 14-day adaptation to diets and 14-day data collection during which feed intake and milk production were recorded daily. Measurements for individual buffaloes were made for water intake, diet, orts, milk, faeces and urine during the final 3 days. The analyses were performed according to OAOC. Lactating buffaloes had greater (P

The domestication of water buffalo likely took place between 4000 and 6000 years ago in the regions of Indus and Yangtze valleys. Domestic water buffalo comprises two subspecies that differ in a number of phenotypic traits, including morphology and behaviour, in geographic range and number of chromosomes. River buffalo has 50 chromosomes and is mainly reared in India, Southwest Asia and the Mediterranean region, including Italy, where the mozzarella di bufala campana cheese is produced from river buffalo milk contributing to the excellent reputation of the made in Italy brand worldwide. Swamp buffalo has 48 chromosomes and is mainly reared in Southeast Asia and China. The river buffalo genome has been recently sequenced and de-novo assembled, through the coordinated effort of an international Consortium led by a core group of Italian scientists. An associated HapMap project has sequenced 47 river buffaloes from 4 breeds at low coverage. As a result, more than 22M SNPs were discovered, permitting the development of the Axiom Buffalo Genotyping 90K (Affymetrix) marker panel. In this study, the SNP panel has been used to assess the main genetic parameters and genetic diversity of different water buffalo breeds sampled in Italy and elsewhere. Genotyping accuracy and main population genetic indexes have been compared between the 90K SNPs from the SNPchip and the 22M SNPs discovered by sequencing. Data were filtered for missing data, MAF ??? 0.05 and deviation from HW equilibrium. Sequence data was filtered for quality score and SNP coverage. Preliminary analyses indicate that genotypes from SNP arrays are better suited to diversity studies than low pass sequence by correctly detecting genotypes at SNP loci. Both sequence and SNP data indicate that expected heterozygosity values are quite variable among breeds. Sequence and SNP array data produced similar estimates of the Fst index (5.2% and 5.6% respectively), revealing that a moderate proportion of the total genetic diversity is explained by the between breed component. Principal Component Analysis showed a clear differentiation among breeds corresponding to geographic origin.

The aim of this work was to evaluate whether L-carnitine supplementation during IVC improves blastocyst development and cryotolerance of in vitro produced (IVP) buffalo embryos. Abattoir-derived cumulus-oocytes complexes (n=410, over 5 replicates) were matured and fertilized in vitro according to standard procedures (Gasparrini B et al. 2006 Theriogenology 65 (2), 275-287). On day 1 (Day 0 = IVF), zygotes were cultured in SOF supplemented with 8 mg/ml BSA, in the absence (control, n=165) or presence of L-carnitine (n=170) at a concentration (0.25 Mm) selected after a preliminary dose response trial. In vitro culture was carried out at 38.5°C under 5% CO2, 7% O2, and 88% N2. Cleavage and blastocyst rates (in relation to the cleaved embryos) were evaluated on Day 5 and 7, respectively. The blastocysts were vitrified by cryotop in 16.5% ethylene glycol, 16.5% DMSO and 0.5M sucrose and the survival rate, based on morphological criteria, was assessed after 24 h culture. Data were analyzed by Chi square test. Cleavage (81.3% vs 82.1%, in the control and carnitine groups) and blastocyst production (40.0% vs 47.6%, in the control and carnitine groups) were not affected by the treatment. The percentages of fast developing embryos (expanded and hatched blastocysts), i.e. those of better quality, were 17.0 and 23.5%, respectively. Interestingly, the embryos cultured with L-carnitine showed higher survival rates after 24 h culture (78.7% and 96.4%, in the control and carnitine groups, respectively; P

The aim was to ascertain whether relationships between corpus luteum (CL) vascularization, CL function, and pregnancy outcome in AI in buffaloes were consistent across the breeding season and transition period to the nonbreeding season in a Mediterranean environment. Stage of the estrous cycle in Italian Mediterranean buffaloes was synchronized using the Ovsynch with timed AI program and buffaloes were mated by AI in both the breeding season (N 131) and transition period (N 125). Detailed investigation of CL structure and function was undertaken in 39 buffaloes at each of the respective times using realtime B-mode/color-Doppler ultrasonography on Days 10 and 20 after AI. Progesterone (P4) concentrations were determined by RIA in all buffaloes. Pregnancy rate on Day 45 after AI was greater (P

The objectives of this work were to evaluate whether the sperm penetration speed is correlated to the in vitro fertility and whether adapting the gamete co-incubation length to the kinetics of the bull improves in vitro fertility and affects the sex ratio. In vitro matured oocytes were co-incubated with spermatozoa from four different bulls (A-D). At various post-insemination (p.i.) times (4, 8, 12, 16 and 20 h), samples of oocytes were fixed and stained with DAPI for nuclei examination, while the remaining ones were transferred into culture to evaluate embryo development. The blastocysts produced were sexed by PCR. Two bulls (A and B) had faster kinetics than the others (C and D), as shown by the higher penetration rates recorded at 4 h p.i. (43%, 30%, 11% and 6%, respectively for bulls A, B, C and D; p

The aim of this work was to compare the efficiency of AI in buffalo in two different periods of the year. The trial was carried out during autumn, i.e. the favorable reproductive season, and midwinter, characterized by a daylight transition towards a positive photoperiod at Italian latitude. In particular, we evaluated if the increase of daily light hours affects early embryonic mortality (EM) rate. Buffalo cows were synchronized by OVSYNCH protocol and artificially inseminated in autumn (n=131) and midwinter (n=125). Twenty-five and 45 days after AI buffaloes underwent ultrasonography. Buffaloes pregnant on day 25 but not on day 45 were considered to have undergone EM. Differences between the two periods were analyzed by Χ-square test. In autumn, pregnancy rate on day 25 after AI was 62.6% and declined to 58.0% by day 45, with an EM rate of 7.3%. In midwinter pregnancy rate on Day 25 after AI was 59.2% and declined to 45.6% by day 45, with an EM rate of 23.0%. The pregnancy rate on day 45 was higher during autumn compared to midwinter (P

The purpose of the review is to describe the factors that affect fertility in domestic water buffalo (Bubalus bubalis) and the techniques that enable an improvement in reproductive performance. On Italian and Latin American farms where natural mating is practiced and bulls are always present in the herd, the inter-calving interval is approximately 400 days and the culling rate is lower than 15%. The buffalo has a tendency for seasonal reproductive activity. Reproduction is favoured when there is a decrease in day length. Ovarian activity stops if conception does not occur within 3 to 5 ovarian cycles. It is important, therefore, that appropriate management of the transition period is practiced, particularly with respect to the hygienic conditions of the uterus. In tropical countries located north of the equator, feed deficiencies and heat stress are considered the main factors that lead to poor fertility in the summer. In Pakistan, for example, the increase in body condition score during the autumn was associated with the commencement of the breeding season in buffaloes. Anoestrus is observed also in Italy, however, where the average daily temperature during the same period is 13.5 to 23.5 degrees C and feeding is constant throughout the year. The only common element between the two areas is the progressive increase in daylight hours between April and June and the day length greater than 12 hours up to September. In Italian herds that apply an out-of-season breeding strategy, an improvement in fertility (measured as the percentage of corpora lutea corresponding to subsequent pregnancy) is observed when water pools are present on the farm. This demonstrates that an improvement in environmental conditions reduces the incidence of embryonic mortality and/or abnormal cycles. To summarize, in the absence of serious nutritional problems, an improvement in environmental conditions increases fertility in buffalo.

The aim in this study was to investigate corpus luteum function and embryonic loss in buffaloes mated by artificial inseminations (AI) during the transitional period from breeding to non-breeding season. The study was carried out using 288 multiparous Italian Mediterranean Buffalo cows at 110 ± 4 days in milk. The buffaloes were mated by AI after synchronization of ovulation by the Ovsynch-TAI protocol 25 days after AI buffaloes underwent trans-rectal ultrasonography to assess embryonic development. Pregnancy diagnosis was confirmed on Days 45 and 70 after AI by rectal palpation. Buffaloes pregnant on Day 25 but not on Day 45 were considered to have undergone late embryonic mortality (LEM), whilst buffaloes pregnant on Day 45 but not on Day 70 were considered to have undergone foetal mortality (FM). Corpus luteum size and blood flow were determined by real-time B-mode/colour-Doppler on day 10 after AI in 122 buffaloes. The resistive index (RI) and pulsatility index (PI) were recorded at the time. Milk samples were collected on Days 10, 20 and 25 after AI in all inseminated buffaloes for the assay of whey P4 concentrations. Data were analysed by anova. Pregnancy rate on Day 25 after AI was 48.6% (140/288) and declined to 35.4% (102/288) and 30.6% (88/288) by Day 45 and Day 70 respectively. The incidences of LEM and FM were respectively 27.1% (38/140) and 13.7% (14/102). Pregnant buffaloes had greater (p

The aim of the work was to evaluate the in vitro developmental competence of in vitro-matured buffalo oocytes after Cryotop vitrification (CTV) and in vitro fertilization (IVF). To optimize parameters, two cryoprotectant (CP) concentrations and two warming-dilution procedures were applied. Oocytes were vitrified in 16.5% ethylene glycol (EG), 16.5% dimethylsulphoxide (DMSO) and 0.5 M sucrose in Groups A and C, and in higher CP concentrations (20% EG, 20% DMSO and 0.5 M sucrose) in Groups B and D. Warming was performed in 1.25 M sucrose for 1 min, then in 0.62, 0.42 and 0.31 M sucrose, 30 s each (Groups A and B), or in 0.25 M sucrose for 1 min and in 0.15 M sucrose for 5 min (Groups C and D). After warming, the oocytes were fertilized and cultured in vitro. Survival rate post-warming was lower in Group D (83.6%) than in Groups A and B (92.4 and 92.8%, respectively), while intermediate values were found in Group C (85.7%). Survival rates at 24 h decreased in Groups C and D (52.0% and 50%, respectively) and remained high in Groups A and B (84.0% and 85.6%, respectively), thus indicating that the dilution of CP after warming is critical for buffalo oocyte cryopreservation. Similar differences were also observed in cleavage rates (42.7%, 55.3%, 28.4% and 36.3% for Groups A, B, C and D, respectively) whereas no differences in blastocyst rates were found among groups (6.4%, 7.8%, 5.9% and 6.9% for Groups A, B, C and D, respectively). Blastocyst production after IVF of vitrified oocytes proves the feasibility of CTV in buffalo species.

Melatonin, the main hormone secreted by the pineal gland, plays many roles in reproduction. In ram spermatozoa, melatonin administration increases plasminogen activator activity (Tsantarliotou MP et al. 2007 Theriogenology 69, 458-465), known to be involved in sperm capacitation and acrosome reaction (Taitzoglou IA et al. 1996 Mol. Androl. 8, 187-197). The aim of this study was to evaluate the efficiency of melatonin to induce buffalo in vitro sperm capacitation, indirectly assessed by estimating the capability of spermatozoa to acrosome-react. Frozen-thawed semen from 4 different bulls was pooled and treated by swim-up in order to select only the motile population. Spermatozoa (n = 829) were assessed immediately after swim- up separation, to evaluate the incidence of acrosomal loss in non-treated cells (time 0). The remaining spermatozoa were incubated in the absence of capacitating agents (negative control; n = 513), in the presence of 0.01 mM heparin (positive control; n = 775), 10 μM melatonin (n = 684), 100 μM melatonin (n = 751), and 1 mM melatonin (n = 650), for 2 h. Sperm were then exposed for 15 min to 60 μg mL-1 of lysophosphatidylcholine, an agent known to induce acrosome reaction (AR) 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 percentage of acrosome-reacted spermatozoa in each group was used to assess the efficiency of capacitation under different incubation conditions. The experiment was repeated 4 times. Differences among groups were analyzed by chi-square test. Acrosomal loss was observed only in 2.1% of the sperm population at time 0. Interestingly, sperm treatment with both heparin and the different concentrations of melatonin resulted in a significantly higher incidence of AR compared to the negative control (24.4, 20.5, 20.0, 23.6 v. 8.0% for the positive control, 10 μM melatonin, 100 μM melatonin, 1 mM melatonin, and the negative control, respectively; P < 0.01). These results demonstrated that melatonin determines capacitation of buffalo spermatozoa in vitro. Furthermore, the effect of melatonin was comparable to that of heparin, that is, the capacitating agent currently used in the IVF system. The capacitating effect was observed at all the tested concentrations, and viability was not affected. This suggests to extend the range of concentrations to test in future studies, in order to identify the optimal dose. Moreover, considering the seasonality of the species and the great differences in fertility attitude of buffalo bulls, it would be interesting to investigate the capacitation effect of melatonin in relation to both the season and the bull.

The aim of this research was to detect possible relations between enzymes activity and sperm function in order to evaluate the potential use of enzymaic tests as markers of sperm quality. Results showed that all enzymes tested are involved in sperm metabolism and their activity is related to sperm quality. Correlation was found in seminal plasma and sperm cells from both fresh and frozen semen thus showing the potential use of enzymati tests to evaluate in vitro sperm fertility

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 vitro fertilization 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% CO2 in 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–1 of OPN; P < 0.01) were observed in the groups with 0.1 and 1 μg mL–1 of 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–1 of OPN; P < 0.01) were observed in the groups with 0.1 and 1 μg mL–1 of 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.