G. M. Terzano

Patterns of oestrus, time of LH release and ovulation and effects of time of artificial insemination in Mediterranean buffalo cows

Thirty-two post-partum Mediterranean river buffalo cows were continuously observed for signs of oestrus from September to December with the aid of two vasectomized bulls. Symptoms of oestrus among female Mediterranean buffaloes are weak, therefore oestrus was assessed based on bull behaviour (following and licking a cow and trying to mount her). Oestrus was divided into three phases based on the bull behaviour assessment. Status of the uterus and ovarian follicles were checked rectally every day for each cow which was detected by a teaser bull to be in oestrus. All cows in oestrus were bred twice by artificial insemination (AI), the first at ovulation and the second (using a different bull) 22 h later. The average duration of interest shown by the bull towards a cow (from the very first to the last sign of interest) was 68 h and the average length of the three phases was: phase 1 = 21 (s.d. 29) h, phase II = 32 (s.d. 24) h and phase III = 15 (s.d. 15) h. Fifteen buffaloes were bled during oestrus, and LH profiles were determined. No differences were evident among oestruses followed by pregnancy (no. = 18) and the others (no. = 26) for the variables describing behavioural events. Neither intensity of the bull courtship, presence or clarity of mucous discharge, or housing system affected the success of AI. The only differences between pregnant and non-pregnant cows were in the timing between the LH peak and the end of phase II (2·4 v. 14·7 h, P v. 40 h, P > 0·05) and ovulation (25 v. 46 h, P

Overview on Reproductive Endocrine Aspects in Buffalo

Buffalo is an important worldwide species in terms of milk and meat production as well as draft. The majorconstraints to full exploitation of the productive potential of buffalo are its inherent low reproductive efficiency due todelayed puberty, higher age at first calving, long post partum anoestrus period, long intercalving period, silent heatcoupled with poor expression of oestrus, seasonality in breeding and low conception rate. Reproductive cycles in buffaloare regulated by endocrine-neuroendocrine interactions between hypothalamic, gonadotropic, gonadal and otherhormones. To improve the buffalo reproductive efficiency, the investigation on endocrine aspects is useful to gain abetter knowledge of buffalo reproduction. This review is focused on the information available on various basic hormones(Melatonin, Progesterone (P4), Estradiol 17B (E2), Follicle stimulating hormone (FSH), Luteinizing hormone (LH)) and onnew hormones (Inhibin, Glycoproteins associated with pregnancy (PAGs), Leptin) related to reproductive function.

Hormonal stimulation and oocyte maturational competence in prepuberal Mediterranean Italian buffaloes (Bubalus bubalis)

The objective of this study was to determine the best combined hormonal treatment to utilize in order to obtain a high number of good quality in vivo and in vitro matured oocytes from prepuberal Mediterranean Italian buffalo calves (Bubalus bubalis). Transvaginal ultrasound follicular aspiration was employed to recover oocytes from antral follicles. Fifteen barn housed buffalo calves, between 5 and 9 months of age were used in this study and randomly divided into control (Group A) and treated groups. A commercially available preparation of 2000 IU eCG was administered to animals in the treatment groups, followed by 2000 IU of hCG given either 12 h (Group B), or 24 h (Group C) before ovum pick up (OPU). From the time of administration of eCG treatments, the best timing for hCG administration before OPU was determined and integrated with the administration of 500 IU of FSH-LH in a decreasing dosage protocol over 4 days (Group D). Expanded cumulus oocyte complexes (COCs) recovered from all groups were immediately fixed for later aceto-orcein staining. All other COCs were processed for in vitro maturation using standard procedures and then fixed and stained for assessment of nuclear maturation. Collectively, hormonal stimulation did not increase the number of ovarian antral follicles available compared to the control group (P > 0.05), but did result in higher output of medium (Group B: 9.8 +/- 7.1; Group C: 3.4 +/- 6.7; Group D: 15.6 +/- 4.9 versus Group A: 1.6 +/- 2.2) and large follicles (Group B: 44.8 +/- 22.9; Group C: 8.7 +/- 6.1; Group D: 70.2 +/- 10 versus Group A: 6.1 +/- 6.3). Administration of hCG 12 h before follicle aspiration proved to be the best strategy to obtain high numbers of immature and mature oocytes from antral follicles (P < 0.05; Group B: 70.8 +/- 12 and Group D: 82 +/- 12.6 versus Group A: 43.6 +/- 13.9 and Group C: 27.2 +/- 13.9). A significantly higher number of expanded COCs was obtained from hormonally stimulated groups compared to the control group (P < 0.05; Group B: 28.7 +/- 16.8, Group C: 16.3 +/- 5.9 and Group D: 27.1 +/- 16.9 versus Group A: 6.2 +/- 6). A higher oocyte maturational competence (P < 0.05) was found in Groups A, B and D (80.8 +/- 7.9, 87.5 +/- 8.2, and 86.5 +/- 4.3, respectively) compared to Group C (60 +/- 26.2). In conclusion, in prepuberal buffalo calves combined gonadotrophin stimulation protocols yielded higher numbers of medium to large size follicles compared to a control group. A high number of good quality oocytes were recovered by transvaginal ultrasound follicle aspiration, and a high rate of metaphase II progression was reached after in vivo and in vitro maturation.

Plasma melatonin in domestic female Mediterranean sheep (Comisana breed) and goats (Maltese and Red Syrian)

The plasma melatonin nychtemeral profiles in Mediterranean ewes and goats were evaluated six times throughout the year. Melatonin levels were high throughout the night and generally below the assay detection limit during daytime. However, during long days, 30% of the last daytime samples had high melatonin concentrations. Plasma melatonin levels were higher in Comisana sheep than in goats, and higher in Maltese than in Red Syrian goats, with highly significant effect of the individual animal and high repeatability. Plasma melatonin was higher in April than in August. When there was a large difference between the duration of day and night, the plasma melatonin pattern and the light/dark cycle did not always match exactly, suggesting some form of superimposition and/or the prevalence of an endogenous rhythm. The difference found at similar scotoperiods with increasing or decreasing day length may be involved in the perception of the photoperiodic changes.

Hormonal dynamics and follicular turnover in prepuberal Mediterranean Italian buffaloes (Bubalus bubalis).

The aim of this study was the investigation of hormonal and ovarian follicular dynamics in prepuberal buffaloes (Bubalus bubalis) bred in Italy. Eleven 5-9-month old buffalo calves ranging in weight from 122 to 270kg, maintained under controlled nutritional and environmental conditions, underwent 50 days of ultrasonographic ovarian follicular monitoring in the months of October-December. Blood sampling for E(2) and FSH determination and ultrasonographic monitoring using a 7.5MHz linear probe and an ALOKA SSD-500 monitor were performed daily. No differences in any of the parameters under study were highlighted when calves were divided into two weight categories (<200 and >200kg) and thus data were pooled. In this study, values are reported as mean+/-S.D. A range of two-six regular follicular waves was reported among calves with an average of 4+/-1.1. Overall interval (days) between wave emergence was 9.9+/-2.8 and largest diameters (mm) of dominant and first subordinate follicles were 8.4+/-1.2 and 4.8+/-0.6, respectively (P<0.05). With the exception of one calf, some minor follicular waves (short waves or SWs; 1.6+/-1), lasting <10 days (6.1+/-1.2) were reported. They were monitored contemporaneously on the ovary contralateral (n=7) or ipsilateral (n=3) to the main follicular wave. Growth rate (mm per day) of dominant follicles (DF) was significantly faster than for corresponding subordinate follicles (SF) and follicles of SWs (1.08+/-0.2 versus 0.79+/-0.1 and 0.83+/-0.1, respectively, P<0.05). The static phase (days) lasted longer in DF compared to SF and SW (5.4+/-1.8 versus 2.4+/-1.2 and 2.6+/-1, respectively, P<0.05). The regressing phase (mm per day) was similar among DF, SF and SW (0.86+/-0.2, 0.94+/-0.2 and 0.84+/-0.1, respectively, P=0.09). Episodic spikes of E(2) and FSH were reported, corresponding to wave development throughout the course of investigation. In conclusion, the majority of buffalo calves displayed a typical pattern of regular follicular development in conjunction with a dynamic trend of ovarian and hypophyseal hormones. Some minor follicle turnover was reported with parallel main follicular waves.

DNA Polymorphisms in River Buffalo Leptin Gene

Abstract Leptin is a protein involved in the regulation of feed intake, fat metabolism, whole body energy balance, reproduction and hematopoiesis. In cattle Leptin gene has been considered a potential QTL influencing several production traits like meat production, milk performance and reproduction. Several studies on bovine leptin gene have found association between polymorphisms and traits like milk yield, feed intake, fat content, carcass and meat quality. With the aim to assess the presence of sequences polymorphisms in the Buffalo leptin gene, we sequenced the entire coding region and part of the introns on a panel of Italian River Buffalos. In this study we identified a new set of SNP (Single Nucleotide Polymorphism) useful for association studies.

LH peak and ovulation after two different estrus synchronization treatments in buffalo cows in the daylight-lengthening period

The aim of this study was to determine the timing of ovulation in relation to the LH peak after synchronization using PRID or Ovsynch protocols, to assess the effects of the period of treatment on these parameters and to provide information concerning how to use the two main protocols for fixed-time artificial insemination in buffalo. Forty-eight lactating Italian Mediterranean buffalo cows were used. The buffaloes were treated in various periods as follows: February to March (n = 12 PRID, n = 12 Ovsynch), end of the breeding season, May to June (n = 12 PRID, n = 12 Ovsynch), beginning of low-breeding season according to Italian environmental conditions. To determine the LH, blood samples were taken at 4-hour intervals, starting 24 hours from PRID removal (PRID group) or 12 hours from (PGF2α) injection (Ovsynch group) up to 108 hours. The ovaries were monitored by transrectal ultrasonography to verify ovulation. The LH-ovulation interval was similar in both groups (30.10 ± 1.05 and 32.77 ± 1.15 hours, respectively, in PRID and Ovsynch group). In the PRID group, the timing of ovulation in relation to device removal was 76.83 ± 3.65 hours with a high level of variability among the animals. In the Ovsynch group, we observed a better synchronization of LH peaks and ovulations, and the timing of ovulation in relation to the last GnRH injection was 35.67 ± 1.15 hours. The percentage of animals reaching the LH peak and ovulation was lower (P ≤ 0.05) in May to June (respectively 75.0% and 54.1%) compared to February to March (respectively 95.8% and 83.3%), indicating a reduction of hypothalamus-pituitary responsiveness to the synchronization treatments in the daylight-lengthening period.

Validation of ELISA kits for determination of Inhibin-A and Estradiol-17-beta concentrations in Buffalo plasma

Abstract The aim was to evaluate the suitability of two commercial ELISA kits for human serum or plasma, to measure Inhibin-A (In-A) and Estradiol-17-beta (E2) concentrations in buffalo plasma. Blood samples were obtained by jugular venipuncture from buffalo heifers and cows, and plasma samples were stored at – 20°C until assays. Precision of the methods was evaluated by the intra- and inter-assay coefficients of variation (CVs) of buffalo plasma sample replicates, at different concentrations. Accuracy was evaluated calculating the recovery rates of different proportions of the highest standard added to a buffalo plasma sample at low concentration (observed/expected values x 100). Linearity was evaluated by serially diluting one buffalo plasma sample at high concentration with the assay buffer and calculating by regression analysis the parallelism of the resulting line with the standard line. Intra-assay CVs were 11% and 15.1% for In-A and 1.8% and 3.3% for E2. Inter-assay CVs were 13.9% and 7.4% for In-A and E2, respectively. Mean recovery rate was 97.9% and 98.5% for In-A and E2, respectively. Dilution tests gave good parallelism between the lines obtained and the standard lines. It is concluded that the kits tested are suitable and reliable for buffalo plasma samples.

Characterization of leptin receptor gene in Bubalus bubalis and association analysis with body measurement traits

Leptin has a pleiotropic effect on regulating appetite, energy metabolism, growth, reproduction, body composition and immunity. This property supports leptin and its receptor as candidate genes for evaluating genetic polymorphisms to associate with growth, milk yield and other economic traits. The aim of this study is to characterize the leptin receptor gene in Bubalus bubalis, to identify single-nucleotide polymorphism (SNP) sites in different coding and non-coding regions and to analyse potential associations between SNPs identified and the body measurements traits of growing buffalo heifers. A group of 64 animals were genotyped by direct sequencing and twenty-eight SNPs were detected. A sequence analysis revealed the presence of nine interesting SNPs in gene sequence. The association analysis of polymorphisms with the body measurements traits of growing buffalo heifers shows significant statistical effects on chest depth and sacrum height. Therefore according to the results obtained from this study, the leptin receptor gene appears to have potential effects on the body measurement traits of Bubalus bubalis.

Follicular development, plasma Inhibin-A and Estradiol-17-beta concentrations in Buffalo cows during different treatment schedules for MOET programs

Abstract Buffalo cows were submitted to three superovulatory treatments. T1 (n = 7): PRID for 10 days (d0-d9) plus decreasing doses of 500 IU FSH/LH (12 h-intervals d7-d10); T2 (n = 8): PRID for 11 d (d0-d10) plus 2000 IU PMSG at d7; T3 (n = 9): PRID for 11 d plus 2000 IU PMSG at d7 and decreasing doses of 175 IU FSH/LH (12 h-intervals d10- d11). Overall plasma inhibin-A (In-A) concentrations correlated with large follicles (LF, diameter >6mm, R=0.83, P<0.01) and small follicles (SF, <6mm, R=-0.21, P<0.01). Plasma E2 correlated weakly with LF (R=0.21, P<0.05). Within treatment, there was always a positive correlation between In-A and LF (T1 R=0.83; T2 R=0.81; T3 R=0.83; P<0.01), but only in T3 there were correlations between In-A and E2 (R=0.67, P<0.01) and SF (R=-0.42, P<0.01), between E2 and LF (R=0.39, P<0.01) and SF (R=-0.35, P<0.05). T3 was followed by a higher number of follicles with diameter >10 mm at d12-13 (T1=5.0+/-1.4, T2=1.2+/-0.9, T3=8.3+/-2.3). In-A concentrations significantly rised at d11-13 of T1 and T3. In-A seems a good indicator of the follicular development during superovulation in buffalo cows, while E2 is not. Furthermore T3 was followed by better ovarian follicular responses.