Cyclic Buffaloes Research Articles

The endocrine changes, the timing of ovulation and the efficacy of the Doublesynch protocol in the Murrah buffalo (Bubalus bubalis)

Experiments were conducted to investigate (a) the timing of ovulation and the associated endocrine changes (progesterone, estrogen and LH) during estrous cycle and (b) the efficacy, with respect to the pregnancy rate, in cycling and anestrus in Murrah buffaloes subjected to the Doublesynch protocol during the low breeding season. In experiment 1, 10 cycling buffaloes were administered PGF2α on day 0 (without regard to the estrous cycle stage), GnRH on day 2, a second PGF2α injection on day 9, and a second GnRH injection on day 11. Transrectal palpation was performed at 2-h intervals after the first and second GnRH treatments until ovulation was detected or for upto 96h. The plasma progesterone and total estrogen concentrations were determined in blood samples collected at daily intervals starting 2days before the onset of the protocol and continued until the day of the second detected ovulation. The plasma LH and total estrogen concentrations were measured in blood samples collected at 30-min intervals for 8h following the first and second GnRH injections and thereafter at 2-h intervals until 2h after the detection of ovulation. Ovulation occurred in 9/10 buffaloes (90%) at 22.2±1.2h (mean±S.E.M.; range 18.0–26.0h) and 10/10 buffaloes (100%) at 23.2±1.0h (mean±S.E.M.; range 20.0–28.0h) after the first and second GnRH treatments, respectively. The peak LH concentrations of 99.8±28.5ng/ml (range 37.8–320.0ng/ml) and 62.3±11.9ng/ml (range 20.9–143.9ng/ml) occurred 2.1±0.3h (range 1.0–3.5h) and 2.3±0.3h (range 0.5–3.0h) after the first and second GnRH treatments, respectively. The total estrogen concentration gradually increased from the day of both the first and second PGF2α administrations until the LH peak (with great variability) and then gradually declined to the basal level, which was reached at the time ovulation was detected. In experiment 2, 10 cycling and 11 non-lactating anestrus buffaloes were subjected to the Doublesynch protocol with timed artificial insemination (TAI) 16 and 24h after the second GnRH treatment, and 55 cycling buffaloes were inseminated after spontaneous estrus was detected (control group). The pregnancy rates were 60% using TAI on cycling buffaloes (experiments 1 and 2), 55% for anestrus buffaloes (experiment 2), and 27.3% for cycling buffaloes inseminated following spontaneous estrus. The overall pregnancy success rates after the Doublesynch protocol in both cycling and anestrus buffaloes increased by 30.8% compared to spontaneous estrus (58.1% vs. 27.3%).In conclusion, the Doublesynch protocol effectively synchronized ovulation twice (after the first and second GnRH treatments) irrespective of the stage of estrous cycle in Murrah buffaloes. The study also demonstrated that the Doublesynch protocol followed by TAI significantly (P<0.005) enhanced the pregnancy rate in cycling and anestrus buffaloes in comparison to untreated controls during the low breeding season.

Isolation and Characterization of Oviduct-specific Glycoproteins from Ampulla and Isthmus Parts of Cyclic and Acyclic Buffalo for Studying Differential Microenvironment

The present study characterized the glycoproteins synthesized by buffalo oviduct. Scanning electron microscopy analyses of the ampullary and isthmic segments of cyclic and acyclic buffaloes showed ultrastructural variations in ciliated and nonciliated cells. Mucosal proteins were extracted by scrapping of different segments of oviduct and, after centrifugation, the remainder tissues were subjected to establish primary cell culture system of oviduct epithelial cells and conditioned media were prepared. Time- and concentration-dependent effects of trypsinization on the establishment of primary monolayer culture showed that 0.25% trypsin for 1-2min at 37°C were the optimal conditions. Total protein content in oviductal tissues and conditioned media was quantified and analyzed by SDS-PAGE which showed marked variation in different segments of the oviduct. Western blot analysis revealed five major oviduct-specific glycoproteins (OGPs) in cyclic oviduct (ampulla and isthmus) with Mw 180, 95, 75, 66 and 35kDa in the oviduct extract and two glycoproteins with Mw 95 and 66kDa in conditioned media. However, in acyclic oviduct (ampulla and isthmus), three glycoproteins were immunostained with Mw 180, 95 and 66kDa in the oviduct extract and one glycoprotein with Mw 66kDa in conditioned media. Indirect Enzyme-Linked Immuno Sorbent Assay (ELISA) results showed significant differences of OGPs in different segments of cyclic and acyclic buffaloes and, thus, indicative of segmental variation in the synthesis and secretion of glycoproteins. Oviductal extract secretes more amounts of OGPs as compared to the conditioned medium. The role of these OGPs may be defined and exploited for influencing the fertilization process and/or subsequent embryonic development.

Antioxidant capacity of follicular fluid in relation to follicular size and stage of estrous cycle in buffaloes

The present study was designed to evaluate the changes in the concentrations of different antioxidants, such as glutathione (GSH), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT), in the follicular fluid collected from different follicular size categories in relation to stage of estrous cycle in buffaloes. In addition, malondialdehyde (MDA) as an indicator for lipid peroxidation was also estimated. Fifty pairs of buffalo ovaries were collected from a local slaughterhouse. Based on ovarian structures, the cycle was divided into follicular and luteal phase. The follicles on each pair were classified into three groups; small (≤3 mm), medium (4–9 mm) and large (≥10 mm). The concentrations of SOD, CAT, GSH, and GR in the follicular fluid of each group as well as MDA were estimated. Results indicated that there was a significant decrease (P < 0.05) in the average numbers of small follicles obtained at the follicular phase than those obtained at the luteal phase of the cycle. However, the mean numbers of the large sized follicles was significantly increased (P < 0.05) in the follicular phase than in the luteal phase. Large follicles obtained at the luteal phase had a significantly higher (P < 0.05) concentration of GSH than that obtained from small ones. A significant (P < 0.05) effect of follicular size on GR concentrations was observed. The concentration of SOD tended to be higher in large follicles obtained at the follicular phase than that collected at the luteal phase (56.7 ± 3.7 vs. 28.1 ± 6.7 U/mL, respectively). On the contrary, a significantly higher concentration (P < 0.05) of SOD was recorded in small follicles as compared with medium and large follicles collected at the luteal phase. CAT concentrations did not significantly differ among different follicular sizes between follicular and luteal phases as well as within each phase. Malondialdehyde concentration was significantly decreased (P < 0.05) in the follicular fluid obtained from small follicles collected at the follicular phase compared with those obtained at the luteal phase. In conclusion, the present study showed that the concentrations of enzymatic antioxidants except for CAT vary according to the follicle size and the stage of the estrous cycle suggesting their possible role in the process of follicular development during estrous cycle in buffaloes.

Alterations in follicular fluid estradiol, progesterone and insulin concentrations during ovarian acyclicity in water buffalo (Bubalus bubalis)

Ovarian acyclicity is one of the most important causes of infertility in water buffalo. Recent studies have indicated alterations in the composition of follicular fluid during the condition. The aim of this study was to determine the changes in follicular fluid concentrations of estradiol, progesterone and insulin during ovarian acyclicity in water buffalo. Ovaries were collected from 50 acyclic and 95 cyclic (control) buffaloes and follicular fluid was aspirated from small (5.0–6.9mm), medium (7.0–9.9mm) and large (≥10.0mm) sized follicles. Estradiol concentration was lower (P<0.0001) in acyclic (1.4±0.09ng/ml) than in cyclic (3.3±0.18ng/ml) buffaloes. Regardless of the ovarian cyclic status, there was an increase (P<0.01) in estradiol concentration with the increase in follicle size; the mean concentrations were 2.4±0.16ng/ml, 2.8±0.29ng/ml and 3.5±0.41ng/ml in small, medium and large follicles, respectively. A higher (P<0.001) progesterone concentration was recorded in acyclic (24.3±2.61ng/ml) compared to the cyclic (7.6±0.79ng/ml) group. Furthermore, acyclic buffaloes had a lower (P<0.05) concentration of insulin in the follicular fluid than that of cyclic buffaloes (15.2±1.55μIU/ml versus 25.9±2.78μIU/ml, respectively). In conclusion, acyclic buffaloes have lower concentrations of estradiol and insulin concurrent with higher concentrations of progesterone in the follicular fluid. These hormonal changes in the follicular microenvironment are possibly a manifestation of the disturbances in the normal follicular development leading to anovulation and anestrus in acyclic buffaloes.

Preovulatory Follicular and Subsequent Luteal Size Influence Pregnancy Success in Water Buffaloes

The diameter of the preovulatory follicle (POF) and its effects on subsequent corpus luteum (CL) size and conception were studied in 38 lactating indigenous cycling buffaloes in the Mymensingh district of Bangladesh. Body condition score (BCS) at estrus was estimated for the buffaloes. The buffaloes were synchronized with two injections of a synthetic analogue of PGF2α administered 11 days apart. Transrectal ultrasonography was carried out at estrus and on days 5, 9, 12 and 16 post ovulation to determine the POF and successive CL size. Pregnancy was confirmed by ultrasound examination on day 40-45 post ovulation. Twenty one (55.3%) buffaloes were diagnosed as pregnant. The conception rates of thin (BCS ≤2.0), good (BCS 2.5-3.5) and fat (BCS glt;3.5) buffaloes were 7.7, 88.2 and 62.5% (χ² = 19.54; P<0.05), respectively. The mean diameter of the POF at estrus was larger (P<0.01) in buffaloes that ultimately were diagnosed as pregnant compared with their nonpregnant counterparts (13.7 ± 0.3 vs. 11.2 ± 0.5 mm, respectively). The conception rates of buffaloes having small (9 to ≤ 12 mm), medium (>12 to ≤14 mm) and large (>14 to 16 mm) POFs at estrus were 9.1, 70.0 and 85.7% (χ² = 13.87, P<0.01), respectively. On day 5 post ovulation, CL size was positively correlated (CL: r=.74, P<0.01) with POF diameter. Retrospective analysis revealed that on day 5 post ovulation, the pregnant buffaloes had higher (P<0.01) post ovulation CL sizes than their nonpregnant counterparts (15.6 vs. 11.8 mm). Similarly, on day 9 post ovulation, the difference in CL size (14.3 vs. 13.6 mm) between pregnant and nonpregnant buffaloes was significant (P<0.05). In conclusion, the diameter of the POF in buffaloes has a positive impact on the size of the post ovulation CL and conception.

228 DOMINANT FOLLICLE ABLATION IMPROVES SUPEROVULATORY RESPONSE AND EMBRYO YIELD IN BUFFALOES SUBJECTED TO INTEGRATED SINGLE-OVULATION EMBRYO TRANSFER - MULTIPLE-OVULATION EMBRYO TRANSFER PROGRAM

Effect of the removal of inhibitory consequences of a dominant follicle (DF) on the recruitment, growth and ovulation of follicles in cyclic buffaloes induced to superovulate, was evaluated. Eight elite, multiparous, donor Murrah buffaloes, 80 to 120 days postpartum, were included in an integrated single-ovulation embryo transfer–multiple-ovulation embryo transfer (SOET-MOET) schedule. Superovulation with FSH (Folltropin® NIH-FSH-P1, Bioniche Animal Health, Belleville, ON, Canada) was preceded by nonsurgical single embryo (SOET) recovery on Day 5 or 6 after prostaglandin F2α (Lutalyse®, Pfizer, Madison, NJ, USA)-induced oestrus and AI (Day 0). For MOET, Folltropin was administered from Day 9 p.m. through to Day 13 p.m. in a twice-daily descending dose schedule (0–5, 5–4, 4–3, 3–2, 2–1 mL; total dose of 580 mg of NIH-FSH-P1). A dose of 25 mg of Lutalyse was administered on Day 12 a.m. and p.m. Donors were inseminated with proven semen on Day 14 a.m. and p.m. and Day 15 a.m. Nonsurgical embryo collection was carried out on Day 19 or 20 followed by administration of Lutalyse on Day 25. Following sexual rest for one complete oestrous cycle, Day 5 or 6 SOET was repeated. On Day 9, ultrasound-guided follicle aspiration was used to ablate the DF and the second MOET treatment was initiated at the same time. Mean diameter of DF at the start of superovulation treatment on Day 9 was similar between the first (12.5 ± 0.66 mm) and second (12.2 ± 0.33 mm) MOET cycles. At AI, mean number (9.6 ± 1.43 vs 13.4 ± 1.87) of follicles &gt;9 mm were higher (P &lt; 0.05) in the second (ablation) MOET cycle, though their mean diameters were similar (11.7 ± 0.21 vs 12.3 ± 0.21 mm; P = 0.17). On the day of ova/embryo collection, almost similar numbers of large follicles (4.1 ± 0.93 and 4.4 ± 0.84), with mean diameters of 12.9 ± 0.74 and 13.4 ± 0.83 mm, were observed for the first and second MOET, respectively. Mean (± SEM) numbers of corpora lutea (3.9 ± 0.81 and 4.9 ± 0.61; P = 0.19) and transferable quality embryos (0.6 ± 0.37 and 1.6 ± 0.37; P = 0.077) were counted in the first and second (ablation) MOET cycles, respectively. These preliminary findings suggest that ablation of DF before start of superovulatory treatment may be beneficial in improving the yield of transferable embryos in a buffalo MOET program. Director, CIRB, is acknowledged for the experimental facilities.

Changes in biochemical composition of follicular fluid during reproductive acyclicity in water buffalo ( Bubalus bubalis)

This study describes the changes in biochemical composition of follicular fluid during reproductive acyclicity in buffalo. A total of 73 pairs of ovaries collected from 26 reproductively acyclic and 47 reproductively cyclic buffaloes were used in the investigation. Ovarian follicles were classified into small (5.0–6.9 mm), medium (7.0–9.9 mm) and large (≥10.0 mm) sized categories depending upon their diameter. Follicular fluid was aspirated, processed and assayed for glucose, cholesterol, total protein, acid phosphatase and alkaline phosphatase. Glucose concentration was lesser in reproductively acyclic compared to cyclic buffaloes (19.3 ± 2.59 mg/dl compared to 32.6 ± 2.60 mg/dl; P < 0.05), mainly due to difference in concentration between small sized follicles (12.4 ± 2.59 mg/dl compared to 28.0 ± 3.32 mg/dl; P < 0.05). Cholesterol concentration was also lesser in reproductively acyclic compared to cyclic buffaloes (32.2 ± 2.14 mg/dl compared to 35.5 ± 2.16 mg/dl; P < 0.05) and this was related to the lesser concentration found in large follicles (13.8 ± 3.45 mg/dl compared to 37.2 ± 4.10 mg/dl; P < 0.001). Total protein and acid phosphatase levels were not affected by either the reproductive cyclicity status or the follicular size (4.9 ± 1.07 g/dl to 6.0 ± 0.28 g/dl and 1.2 ± 0.17 U/dl to 2.5 ± 1.22 U/dl, respectively). An increased alkaline phosphatase activity was, however, observed in reproductively acyclic compared to cyclic buffaloes (27.5 ± 3.08 U/dl compared to 14.0 ± 1.09 U/dl; P < 0.0001). In conclusion, results of the present study indicate an alteration in the biochemical composition of follicular fluid during reproductive acyclicity in buffalo. The findings provide further support to the notion that poor nutrition is an important factor triggering reproductive acyclicity in buffalo.

Follicular characteristics and intrafollicular concentrations of nitric oxide and ascorbic acid during ovarian acyclicity in water buffalo (Bubalus bubalis)

The objective of this study was to examine the follicular characteristics and intrafollicular concentrations of nitric oxide and ascorbic acid during ovarian acyclicity in buffaloes. Ovaries were collected from 56 acyclic and 95 cyclic buffaloes at slaughter, surface follicle number was counted and follicles were classified into small (5.0-6.9 mm), medium (7.0-9.9 mm), and large (≥ 10.0 mm) size categories based on their diameter. Follicular fluid was aspirated and assayed for nitric oxide, ascorbic acid, estradiol, and progesterone. Acyclic buffaloes had a higher (P<0.05) number of medium-sized follicles and a lower (P<0.001) number of large follicles than the cyclic ones. In acyclic animals, the number of large follicles was lower (P<0.01) than in medium size category which in turn was lower (P<0.001) than the number of small follicles. In contrast, the number of medium and large follicles was not different (P>0.05) in the cyclic control. However, the number of small-sized follicles was higher (P<0.001) compared to the other two categories. The incidence of large-sized follicles was lower (P<0.05) in acyclic buffalo population compared to the cyclic control. Evaluation of estrogenic status demonstrated that all the follicles of acyclic buffaloes are estrogen-inactive (E (2)/P (4) ratio<1). Small- and medium-sized follicles of acyclic buffaloes had higher concentrations of nitric oxide (P<0.05 and P<0.001, respectively) and lower concentrations of ascorbic acid (P<0.05 and P<0.01, respectively) than the corresponding size estrogen-active follicles of their cyclic counterparts. In conclusion, this study indicates that follicular development continues during acyclicity in buffaloes. Although follicles in some acyclic buffaloes attain a size corresponding to morphological dominance, they are unable to achieve functional dominance, perhaps due to an altered balance of intrafollicular nitric oxide and ascorbic acid and, as a result, these follicles instead of progressing to ovulation undergo atresia.

Follicular fluid nitric oxide and ascorbic acid concentrations in relation to follicle size, functional status and stage of estrous cycle in buffalo

The objective of this study was to evaluate the changes in follicular fluid nitric oxide (NO) and ascorbic acid (AA) concentrations with varying follicle size and functional status, and stage of estrous cycle in buffalo. The main effect of follicle size on NO concentration and size-by-status interaction were statistically significant ( P < 0.05). Small follicles had a higher ( P < 0.05) NO concentration compared to medium and large follicles. Further, estrogen-active (EA) small follicles showed increased ( P < 0.05) NO concentrations than the corresponding estrogen-inactive (EI) follicles. Within EA category, higher ( P < 0.01) concentrations were recorded in small compared to medium and large follicles. There was no significant main effect of stage ( P > 0.1) on NO concentration but the stage-by-size interaction was significant ( P < 0.05) with medium follicles showing a higher ( P < 0.05) concentration during late luteal stage compared to the mid luteal stage. During early and mid luteal stages, higher ( P < 0.05) NO concentrations were recorded in small than in medium follicles. A significant ( P < 0.01) main effect of size on AA concentration was observed with higher values in medium than in small and large follicles. Size-by-status interaction for AA approached statistical significance ( P < 0.06) with higher ( P < 0.05) concentrations recorded in medium than in large EA follicles. The main effect of stage on AA concentration was, however, non-significant ( P > 0.1) but the stage-by-size interaction approached statistical significance ( P < 0.06) with lower ( P < 0.05) levels recorded in large compared to medium size follicles during the follicular stage. In conclusion, the present study demonstrates that the concentrations of follicular fluid NO and AA vary according to the follicle size, functional status and stage of estrous cycle suggesting their possible role in process of follicular development during estrous cycle in buffalo.

Impact of pre-ovulatory follicle diameter on plasma estradiol, subsequent luteal profiles and conception rate in buffalo (Bubalus bubalis)

The present study was designed to investigate the impact of pre-ovulatory follicle (POF) diameter on the day of estrus on plasma estradiol concentration, subsequent luteal profile (corpus luteum, CL, diameter and plasma progesterone concentration) and conception rate in buffaloes. Twenty-eight buffaloes were synchronized with synthetic analogue of prostaglandin F2α (PGF2α) administered 11 days apart. Transrectal ultrasonography and jugular vein blood sampling was carried out on the day of estrus and on days 0 (day of ovulation), 5, 12, 16 and 21 post-ovulation. Out of 28 buffaloes, 11 (39.3%) were diagnosed pregnant on day 40 post-ovulation. Retrospective analysis revealed that the buffaloes getting pregnant had larger (p<0.05) POF diameter. In fact, all the buffaloes (n=5/5) having POF diameter between >14 and 16mm conceived, whereas, conception rate in buffaloes with POF diameter between >12 and ≤14mm (n=6/17) or <12mm (n=0/6) was 35.3% and 0.0%, respectively. A positive correlation (r=0.57, p<0.05) was observed between POF diameter and plasma estradiol concentration at estrus. On day 5 post-ovulation, luteal profile was positively correlated (CL: r=0.34, p<0.05; plasma progesterone concentration: r=0.27, p>0.05) with POF diameter. Further, on the same day, plasma progesterone concentration was positively correlated (r=0.47, p<0.05) with CL diameter, however, this correlation was absent (r=0.05, p>0.05) during the subsequent luteal phase. Nevertheless, the post-ovulation luteal profile of pregnant buffaloes was higher (p<0.05) compared to non-pregnant counterparts. In conclusion, the diameter of POF in buffaloes has positive impact on plasma estradiol concentration at estrus, post-ovulation luteal profile and conception rate. The diameter of CL can be used as an indicator of luteal function at early but not at mid or late luteal phase of estrus cycle in buffaloes.

Bilateral follicular cysts in a water buffalo

The present short communication puts on record a case of bilateral, multiple follicular cysts in a water buffalo along with a detailed description of its ovarian biometry and follicular fluid composition. The ovarian weight and biometrical parameters were much higher than in normal cycling buffaloes. A total of three follicular cysts were observed, two on the right ovary and one on the left ovary, measuring 4.9, 3.0 and 2.6 cm yielding 21, 9 and 5 ml of follicular fluid, respectively. The cystic fluid was deep yellow in colour with a viscous consistency. The follicular fluid concentrations of glucose, total protein, cholesterol, acid phosphatase, calcium, phosphorus and progesterone in all the cysts were within the range reported previously in normal buffalo follicular fluid; however, the alkaline phosphatase concentration in cyst 1 and total bilirubin concentration in cysts 1 and 2 were higher than the values in normal follicular fluid. In contrast, the levels of urea nitrogen in cysts 1 and 3, and oestradiol in cyst 3 were lower than the normal values. All the three follicles had an oestradiol to progesterone ratio less than 1. The results of our study suggest that follicular cysts in buffalo are oestrogenically inactive and have an altered concentration of certain biochemical and hormonal constituents.

Fixed-time artificial insemination in buffalo

Artificial insemination (AI) in buffalo has limited use worldwide due the difficulties in the estrus detection and in finding an adequate moment for this procedure. Therefore, an alternative to increase the number of buffalo that are inseminated is the use of protocols that allow the AI without the need of estrus detection, usually called fixed-time AI (FTAI). Our expanding knowledge of the control of follicular wave dynamics during the buffalo estrous cycle has resulted in renewed enthusiasm for the prospects of precisely control of the follicular and luteal dynamics and finely controlling the time of ovulation. Follicular wave development can be controlled by treatments with GnRH or estradiol and progestogen/progesterone in combination. Treatment of buffalo with GnRH in combination with prostaglandin F2α (PGF2α) 7 d later and a second GnRH 48 h after PGF2α (known as Ovsynch) has resulted in acceptable pregnancy rates after FTAI in cycling buffalo during the breeding season. FTAI protocols using progestin devices, estradiol and eCG have resulted in synchronous onset of a new follicular wave, synchronous ovulation and consistent pregnancy rates in anestrous buffalo during the off breeding season. The combination of these protocols permits the use of AI throughout the year, obtaining conception and calving even in anestrus buffalo during the off breeding season.

Endocrine Profiles of Oestrous Cycle in Buffalo: A Meta-analysis

A meta-analysis was conducted to summarize the results of studies which have described the profiles of hormones during the oestrous cycle in buffalo using a fixed effect model and a random effect model. Plasma progesterone concentrations were lowest (0.30±0.06 ng/ml) during the peri-oestrous phase and increased (p = 0.067) through the early luteal phase to a maximum concentration (1.94±0.03 ng/ml) during the mid-luteal phase. Circulating plasma inhibin and estradiol concentrations were lowest (0.31±0.01 and 11.04±0.13 ng/ml) during the mid-luteal phase, increased through the late luteal phase to maximum concentrations (0.44±0.02 and 22.48±0.32 ng/ml) during the peri-oestrous phase. Plasma FSH concentrations were lowest during the early luteal phase and increased through the mid-luteal phase to a maximum concentration during the peri-oestrous phase. Peripheral prolactin concentrations were lowest during the late luteal phase and increased to a maximum concentration during the peri-oestrous phase which then declined (p = 0.716) during the early luteal phase. Peripheral plasma cortisol concentrations decreased from 2.68±0.14 ng/ml during the early luteal phase to 1.43±0.27 ng/ml during the mid-luteal phase (p<0.001) which then increased to 2.06±0.17 ng/ml during the late luteal phase. Plasma T 3 concentrations decreased from the late luteal phase to the peri-oestrous phase (p<0.001) which then increased during the early luteal phase. T 4 concentrations increased from the late luteal phase to the peri-oestrous phase which then decreased during the early luteal phase.

Ion, Protein, Phospholipid and Energy Substrate Content of Oviduct Fluid During the Oestrous Cycle of Buffalo (Bubalus bubalis)

The aim of this research was to analyse the composition of oviduct fluid (ODF) in buffalo cows at different oestrous cycle phases to fulfil the requirements of buffalo embryos in vitro. ODF was collected by chronic cannulation from three cows that were synchronized by administering a synthetic prostaglandin. Based on hormonal profiles, the pre-ovulatory, ovulatory, post-ovulatory and luteal phases of the oestrous cycle were defined. The volume of ODF produced (ml/24 h) was influenced by the oestrous cycle, with values (mean ± SE) around ovulation (1.0 ± 0.2) greater (p < 0.05) than in both the luteal (0.4 ± 0.1) and the post-ovulatory phases (0.5 ± 0.1), but not different from the intermediate values in the pre-ovulatory phase (0.8 ± 0.2). Among cycle phases, no differences were found in sodium, potassium, calcium and magnesium concentrations (130.0 ± 1.1, 5.1 ± 0.3, 2.8 ± 0.1 and 0.59 ± 0.04 mmol/l respectively). Interestingly, the chloride secretion (μm/24 h) was higher (p < 0.05) at ovulation (150.2 ± 16.5) than during both the luteal (73.7 ± 22.0) and the post-ovulatory phases (63.7 ± 11.2), with intermediate values in the pre-ovulatory phase (113.4 ± 23.5). Glucose concentration (mmol/l) was higher (p = 0.056) in the pre-ovulatory phase (0.06 ± 0.02) than in the luteal (0.02 ± 0.01) and post-ovulatory (0.02 ± 0.01) phases but not different from values in the ovulatory phase (0.04 ± 0.02). Concentrations of pyruvate and lactate among oestrous cycle phases were similar (0.08 ± 0.01 and 1.0 ± 0.1 mmol/l respectively). The total quantity of phospholipids (μmol/24 h) was greater (p < 0.05) at ovulation (0.21 ± 0.02) compared with the luteal, pre-ovulatory and post-ovulatory phases of the cycle (0.09 ± 0.02, 0.13 ± 0.02 and 0.09 ± 0.01 respectively). No differences were found in either the protein concentration (1.8 ± 0.3 mg/ml) or the quantity of proteins secreted in 24 h (1.8 ± 0.4 mg) among oestrous cycle phases. In conclusion, this study provides the first characterization of buffalo ODF during the oestrous cycle, showing species-specific differences that may be useful for developing suitable media for buffalo in vitro embryo production.

Relationship between peripheral plasma inhibin and FSH concentrations in Sahiwal cows (Bos indicus) and Murrah buffaloes (Bubalus bubalis) during estrous cycle

The present investigation was undertaken to study the relationship between circulating inhibin and FSH concentrations during the estrous cycle in buffaloes and Sahiwal cattle. The pattern of inhibin concentrations was similar, with peak concentrations on Day -2 (Day 0 = day of estrus) and minimum concentrations on Days 12 and 11 in buffaloes and cattle, respectively. Circulating FSH concentrations were the highest on Day 0 and lowest on Days 8 and 13 in buffaloes and cattle, respectively. Peripheral plasma inhibin concentrations were negatively correlated to FSH concentrations in buffaloes (r = -0.27, P < 0.01) and cattle (r = -0.35, P < 0.01) indicating that inhibin is involved in negative feedback regulation of FSH in both these species.

Superovulatory response of water buffalo (Bubalus bubalis) to gonadotrophins out of the breeding season

Ten adult cyclic female buffaloes (5 normo-karyotype and 5 hybrids) were treated with a conventional superovulation protocol following 2 luteolytic doses (25 mg, i.m.) of PGF2α 11 days apart (days -11 and 0). In all cases, ovulation post PG treatment was confirmed by ultrasound. Superovulation was initiated on d 12 (~d 10 of cycle) with FSH administered twice daily in decreasing doses/day. Preliminary results obtained here suggest failure in ovulation after the superovulation treatment with FSH in water buffalo during the non breeding season, although the follicular recruitment and further development of ovulatory follicles was evident.

Follicular dynamics during synchronization of ovulation of nuliparous buffaloes cows during unfavourable reproductive station

The experiment was conduct to assess the follicular dynamics, time and synchrony of ovulation in nuliparous Murrah buffaloes (Bubalus bubalis) treated with an ovsynch protocol associated with progesterone. Eight nuliparous cycling buffaloes received an intravaginal CIDR device containing 1.9 g of progesterone and 100 mg of a GnRH analogue i.m. (Gonadorelin, Profertil) without regard to the stage of the estrous cycle (day of treatment, day 0) followed by 500 UI eCG (eCG, Novormon) 7 days later and 0.150 mg of PGF2α i.m. (d-Cloprostenol, Prolise) 8 days later at the time of CIDR device removal. A second-treatment of the same GnRH analogue (100 mg, i.m.) was given 48 h after PGF2α. All buffaloes presented a BSC of 3,5 (scale 1 to 5, 1 = very thin and 5 = fat) Follicular dynamics was monitored by transrectal ultrasound (Pie Medical, linear transducer of 7.5 MHz ). Only one female ovulated after first GnRH application. The diameter of the ovulatory follicle at the day of device removal was 7.5 ± 1.0 mm and at the day of second GnRH injection was 10.5 ± 1.2 mm. The interval between device removal and ovulation was 67.0 ± 6.0 hours and ovulation rate was 75% (6/8).

Status of Lipid Peroxidation in Normal Cycling and 慣-Tocopherol Supplemented Anestrus Buffalo Heifers (Bubalus bubalis)

The present study was undertaken to analyze the status of erythrocytic malonyl dialdehyde (MDA) as an index of lipid peroxidation in normal cycling and α-tocopherol supplemented anestrus buffalo heifers. Before supplementation, the blood samples were collected at weekly interval in anestrus and normal cycling buffalo heifers and data from four samples was pooled to establish pre-supplementation base line. Thereafter, the blood samples from anestrus and α-tocopherol supplemented anestrus buffalo heifers were taken at fortnightly interval for 12 weeks during supplementation and for four weeks during the post-supplementation period. The pre-supplementation endogenous erythrocytic MDA, 0.3% H 2 O 2 induced-erythrocytic MDA and 1.5% H 2 O 2 induced-erythrocytic MDA levels (nmol/g Hb) increased significantly (p<0.05) in anestrus heifers (372.3±5.0, 564.4±5.7, 862.4±8.5) when compared to normal cycling animals (289.6±3.0, 508.2±6.0, 777.7±4.9). Oral supplementation of 3,000 mg of a-tocopherol per week per animal in anestrus heifers resulted in a progressive and significant (p<0.01) decline in erythrocytic MDA levels. These observations implied that supplementation of α-tocopherol to anestrus buffalo heifers ameliorated their antioxidant status by alleviating the effects of oxidative stress.

Effect of Removal of Follicles through Repeated Transvaginal Follicle Aspiration on Subsequent Follicular Populations in Murrah Buffalo (Bubalus bubalis)

This study was conducted to investigate the effects of removal of all ovarian follicles through repeated transvaginal follicle aspiration (TVFA) on the subsequent follicular populations in buffaloes. This information is crucial for determining the optimum time interval between successive aspirations for recovering oocytes from live buffaloes through Transvaginal Oocyte Retrieval (TVOR). The oestrus of cycling buffaloes (n=5) were synchronized by a single PGF injection schedule. All the ovarian follicles were removed once every 7 days for 6 weeks through TVFA, starting from Day 7 of the oestrous cycle (Day 0 = day of oestrus). The number and size of individual ovarian follicles was recorded at Day 3 and Day 5 (Day 0 = day of TVFA) through transrectal ultrasonography. The follicles were classified on the basis of their diameter as small (3-5 mm), medium (6-9 mm) and large (≥10 mm). There was no difference in the number of small and medium follicles, and the number of total follicles between Day 3 and Day 5. However, the number of large follicles was significantly higher (p<0.05) at Day 5 than that at Day 3. There was a significant (p<0.05) decrease in the proportion of small follicles and an increase (p<0.05) in the proportion of large follicles from Day 3 to Day 5, with no change in the proportion of medium follicles. The number of total follicles at Day 3 or Day 5 did not differ during the 6 TVFA sessions. It can be concluded that an interval of 3 days is more suitable than that of 5 days between successive aspirations for recovering oocytes through TVOR in a twice weekly schedule and that repeated removal of follicles through TVFA does not adversely affect the number of total follicles 3 or 5 days after TVFA. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No. 5 : 632-636)

Changes in uterine protein secretion during luteal and follicular phases and detection of phosphatases during luteal phase of estrous cycle in buffaloes ( Bubalus bubalis)

Changes in uterine proteins during different reproductive states and their functional significance though known in other species have not been established in buffaloes. An attempt has been made to unravel the changes in composition of buffalo uterine secretion with growth and regression of corpora-lutea during early, mid and late luteal and follicular phase of estrous cycle using gel filtration and electrophoresis techniques. Also the phosphatases activities in luteal phase uterine secretions have been studied. Gel filtration chromatography analysis revealed a protein peak in void volume of the column, the intensity of which was more in all the luteal phase samples than follicular phase samples. Alkaline phosphatase was also found eluted in the void volume. The other three uterus-specific peaks (Peaks V–VII) were detected below 13.7 kd molecular weight. There were at least five peaks of acid phosphatases activity in chromatogram. Silver staining of SDS-PAGE gel detected as many as 40 protein bands in the uterine fluid of which nine proteins were glycoproteins. Molecular weight (MW) comparison revealed the major protein band at 66 kd which could be serum albumin. Comparison of uterine proteins with serum protein bands revealed a 93.5 kd glycoprotein in buffalo serum that did not appear in uterine fluid and at least 11 uterus-specific protein bands (506, 470, 241, 114, 49, 38, 33, 26, 19.2, 16, and 14.3 kd). The 38 and 19.2 kd bands were luteal-stage specific. Intense periodic acid Schiff's (PAS) stained bands in uterine proteins compared to serum indicated glycosylation process in endometrial epithelial cells. The study suggested that buffalo uterine secretion contained mainly serum and several uterus-specific proteins of which few were luteal phase specific. Further study on characterizing the unique or most abundant proteins and defining their role in uterine functions would help to address the cause of low reproduction rate in buffaloes.