Semen characteristics of the swamp buffalo (Bubalus bubalis)

The semen characteristics were studied in 182 ejaculates collected with a bovine artificial vagina from five swamp buffalo (Bubalus bubalis) bulls. The mean values were: volume, 2.9 ml; general motility, 70.7%; live (unstained) sperm, 86.5%; abnormal sperm, 10.3%; intact acrosomes, 82.4%; sperm concentration, 1.06 × 10⁹cells/ml and total sperm/ejaculate, 3.18 × 10⁹cells/ml. Among the sperm abnormalities noted were “knobbed” acrosome, abaxial implantation, the “Dag” defect and the corkscrew midpiece. There were no significant (P > 0.05) monthly variations for any of the semen characteristics studied.     

Cryopreservation of buffalo (Bubalus bubalis) semen in Bioxcell extender

This study was designed to compare commercially available extender Bioxcell^® with tris-citric egg yolk extender for post thaw quality and in vivo fertility of buffalo semen. For comparison of post thaw semen quality: semen was collected from five adult Nili-Ravi buffalo (Bubalus bubalis) bulls of similar age group with artificial vagina (at 42 °C) for three weeks (replicates). Qualifying ejaculates having motility >60% from each buffalo bull were divided in two aliquots and diluted (at 37 °C having 50 × 10⁶ spermatozoa/ml) in tris-citric egg yolk or Bioxcell^® extender. Diluted semen was cooled to 4 °C in 2 hours, equilibrated for 4 hours and filled in 0.5 ml straws. Semen straws were kept over liquid nitrogen vapors (5 cm) for 10 minutes. Straws were then plunged and stored in liquid nitrogen (−196 °C). After 24 hours of storage, semen straws were thawed at 37 °C for 30 seconds to assess sperm motility, viability, plasma membrane integrity, normal apical ridge, and abnormalities (head, mid piece, and tail). For comparison of in vivo fertility: semen from two buffalo bulls of known fertility was cryopreserved in tris-citric egg yolk and Bioxcell^® as described earlier, and used for inseminations under field conditions. Post-thaw percentage of sperm motility (45.3 ± 1.1, 45.0 ± 1.4), viability (66.2 ± 1.1, 64.4 ± 1.3) plasma membrane integrity (60.4 ± 1.2, 59.2 ± 1.4) and normal apical ridge (82.9 ± 0.5, 80.7 ± 0.5) did not differ (P > 0.05) in tris-citric egg yolk and Bioxcell^® extender, respectively. Similarly, sperm abnormalities of head (1.20 ± 0.1, 1.20 ± 0.1), mid piece (0.67 ± 0.1, 0.87 ± 0.1) and tail (11.7 ± 0.2, 11.6 ± 0.3) remained similar (P > 0.05) in tris-citric egg yolk and Bioxcell^® extender, respectively. In vivo fertility rates of buffalo semen cryopreserved in tris-citric egg yolk and Bioxcell^® also remained similar (44% vs. 47%). It is concluded that commercially available Bioxcell^® may be used for the cryopreservation of buffalo semen with an equal efficiency to tris-citric egg yolk extender.     

Glutathione-S-transferase: Role in buffalo (Bubalus bubalis) sperm capacitation and cryopreservation

In this study, glutathione-S-transferase Mu3 (GST) has been reported to play an important role in sperm capacitation, acrosome reaction, and fertilization. The freshly ejaculated buffalo spermatozoa were in vitro capacitated using heparin (10 μg/mL) or cryopreserved in egg yolk citrate extender. Glutathione-S-transferase was identified and characterized in terms of their isozymic forms, tyrosine phosphorylation, and immunolocalization patterns in cryopreserved buffalo spermatozoa in comparison with freshly ejaculated and in vitro capacitated spermatozoa. Two-dimensional gel electrophoresis, immunoblot, immunocytochemistry, and enzyme activity analyses were done to characterize GST in this study. Five and eight isozymic forms of GST were detected in cryopreserved and capacitated spermatozoa, respectively. Differential tyrosine phosphorylation of these enzymes was observed in cryopreserved and capacitated spermatozoa. The tyrosine phosphorylation of this enzyme involved cAMP protein kinase-A dependent and extracellular signal-regulated kinase independent pathways during in vitro capacitation of the spermatozoa. Differential immunolocalization patterns of GST were observed in freshly ejaculated, capacitated, and cryopreserved spermatozoa. Glutathione-S-transferase Mu3 enzyme activity was found to be significantly (P < 0.05) different in freshly ejaculated, capacitated, and cryopreserved spermatozoa. Activity of GST was significantly (P < 0.05) increased with the progression of capacitation. The cryopreserved spermatozoa showed significantly (P < 0.05) greater enzyme activity compared with fresh spermatozoa and was equal to 2-hour capacitated spermatozoa. The cryopreserved spermatozoa showed significant (P < 0.05) loss of GST enzyme protein. Tyrosine phosphorylated GST showed significantly (P < 0.05) greater activity compared with their dephosphorylated forms. The information generated in this study can be used to understand the molecular mechanism of the effects of GST on capacitation. Regulation of GST during sperm cryopreservation could be a good target to improve fertility of cryopreserved spermatozoa for their use in assisted reproductive technologies.     

Gas chromatographic-mass spectrometric analysis of chemical volatiles in buffalo (Bubalus bubalis) urine

Isolation of active fraction and characterization of chemosignals from urine have been attempted in several mammalian species in the recent years. The objective of this study was to identify the urinary volatiles across various reproductive stages of buffalo cow, namely, estrus, diestrus, and pregnancy, and in bull, by chemical extraction followed by gas chromatography–linked mass spectrometry (GC-MS). Urine samples were collected from six buffalo cows at two different phases of estrous cycle, namely, estrus and diestrus. Besides, urinary samples were collected from five pregnant buffalo cows (60–75 days after artificial insemination (AI)) and six adult bulls. Thin-layer chromatography was performed as a preliminary test for qualitative comparison of different compounds extracted by organic solvents. Identification of the urinary compounds was carried out in a gas chromatograph (Perkin Elmer, Autosystem XL) linked to a mass spectrometer (Turbomass). The results of GC-MS analysis indicated the presence of 21 compounds with varying molecular weights and retention time, which were further categorized as diestrus-specific, pregnancy-specific, and bull-specific urinary compounds. No compound, however, could be identified as estrus-specific. We concluded that qualitative differences do exist in estrus, diestrus, and pregnant buffalo cow urine and in bull urine, as evidenced by GC-MS.     

Oestrus synchronization in buffaloes (Bubalus bubalis)

A study was conducted on 34 Surti buffalo cows to determine the feasibility of synchronizing oestrus using prostaglandin F_2α and a 12-day progesterone intravaginal device. Eighteen cycling buffalo cows having palpable corpora lutea were treated with a single intramuscular injection of 30 mg of prostaglandin F_2α. Three cows exhibited oestrus approximately 54 h after treatment and two of these were diagnosed pregnant 90 days after natural breeding. Sixteen randomly selected post partum cows were treated for 12 days with a progesterone intravaginal device. Ten mg of oestradiol 17β in 5 ml of ether was also injected at the time of insertion of the device. Thirteen cows retained the device for 12 days and 10 of them returned to oestrus 4–5 days after its removal. Eight animals were diagnosed pregnant 90 days after natural breeding. The results indicate that short term progesterone intravaginal device treatment is more reliable than prostaglandin for synchronizing oestrus in buffaloes.     

Post partum ovarian activity and uterine involution in the suckled swamp buffalo (Bubalus bubalis)

Ovarian activity and uterine involution were monitored by rectal palpation, oestrus detection and plasma progesterone analysis from 3 to 4 days to approximately 150 days post partum in 38 suckled swamp buffaloes (Bubalus bubalis). The intervals from parturition to regression of the corpus luteum (CL) of pregnancy and involution of the uterus were 10 and 28 ± 6 (S.D.) days respectively. First detected oestrus and first elevation of plasma progesterone (> 0.7 ng/ml) occurred at 88 ± 26 and 96 ± 22 days in 8 and 12 buffaloes respectively. During the first 150 days post partum, 26 of 38 suckling buffaloes (68%) were acyclic (anoestrus) and of 12 animals (32%) exhibiting ovarian cycles, 4 were not detected in oestrus. The tentative diagnosis, based on rectal palpation, that CL were present between days 30 and 90 after parturition (without concurrent luteal levels of progesterone in plasma) suggests that confirmation should be by laparoscopy. It is concluded that a delay in the resumption of ovarian cyclicity post partum represents an important factor contributing to the prolonged calving to conception interval in the suckled swamp buffalo.     

Pregnancies established from handmade cloned blastocysts reconstructed using skin fibroblasts in buffalo (Bubalus bubalis)

Handmade cloning (HMC), a simple, micromanipulation-free cloning technique, has been applied for the production of cloned embryos and offspring in many livestock species. The objective of the present study was to compare the effect of donor cell type on developmental competence of HMC embryos and to explore the possibility of establishing pregnancies using these embryos in buffalo. After technical optimization of the HMC procedure for in vitro development of cloned blastocysts, various donor cells were compared for their developmental efficiency. Using buffalo fetal-, newborn-, adult fibroblasts and cumulus cells, blastocyst production rates obtained from reconstructed embryos were 24.0 ± 1.8% (35/145), 33.0 ± 8.0% (56/163), 21.0 ± 9.3% (29/133) and 49.6 ± 1.9% (77/154), respectively. Blastocyst rates were higher (P < 0.05) in cumulus cell reconstructed embryos in comparison to those derived from fetal or adult fibroblasts. Pregnancy diagnosis (transrectal ultrasonography) was carried out at Day 40 of gestation. Following transfer of HMC embryos reconstructed using newborn fibroblasts 25% (2/8) buffaloes were pregnant and are at Days 201 and 94 of gestation, whereas after transfer of HMC embryos reconstructed using fetal fibroblasts, 20% (1/5) buffaloes were pregnant and are at Day 73 of gestation. In conclusion, HMC could be a simple and efficient technique for the production of cloned embryos for establishing pregnancies in buffalo.     

Cryogenic changes in proteases and antiprotease activities of buffalo (Bubalus bubalis) and cattle (Bos taurus) semen

The postthaw motility and fertility of buffalo and cattle semen is reduced when they are cryopreserved for artificial insemination. In the present study, an attempt was made to characterize the cryogenic changes in proteases and antiprotease activities (APA) of buffalo and cattle semen because these proteolysis regulators have been reported to be associated with sperm motility and fertility. Buffalo sperm demonstrated at least two major proteases of 45 and 42 kDa and three minor proteases of 95, 52, and 33 kDa. Similarly, cattle sperm demonstrated three major proteases of 62, 45, and 42 kDa and two minor proteases of 85 and 78 kDa. Buffalo seminal plasma demonstrated at least three major proteases of 78, 68, and 62 kDa and one minor protease of 98 kDa and cattle seminal plasma demonstrated one major protease of 68 kDa and two minor proteases of 78 and 75 kDa. Except for the 45 kDa protease, most of the previously mentioned proteases were found to be metalloproteinases. Compared with fresh sperm, cryopreserved buffalo and cattle sperm demonstrated a major protease band of 52/49 kDa and the activity of this protease reduced progressively with the duration of cryopreservation. On the contrary, compared with the fresh seminal plasma, cryopreserved buffalo and cattle semen extenders displayed the presence of a new protease band of 45 kDa and demonstrated that this protease activity was leaked from buffalo and cattle cryopreserved spermatozoa. Buffalo and cattle seminal plasmas displayed at least two major APA of 86 and 26 kDa. Compared with buffalo, cattle seminal plasma demonstrated significantly greater APA. Thus, the present study demonstrated the presence of an array of proteases and APA in buffalo and cattle semen and the activities of which changed during cryopreservation. The leakage of the specific protease activity and changes in the proteases and APA might be attributed to reduced motility and fertility of cryopreserved semen in these species.     

Butylated hydroxytoluene inclusion in semen extender improves the post-thawed semen quality of Nili-Ravi buffalo (Bubalus bubalis)

The study was carried out to evaluate the potential impact of butylated hydroxytoluene (BHT) on the frozen-thawed semen quality of Nili-Ravi buffalo bulls. Ejaculated bull semen was extended in a Tris-citrate egg yolk extender containing various concentrations of BHT (0.5, 1.0, 2.0 and 3.0 mM). Semen was frozen at −196 °C using 50 × 10⁶ spermatozoa per 0.5 mL straws. Five straws from each treatment were thawed to assess the semen quality in terms of sperm motility, viability, plasma membrane integrity and acrosomal integrity. Post-thawed sperm motility was determined using a phase-contrast microscope. Viability, plasma membrane integrity and acrosomal integrity were evaluated by the supravital staining, hypo-osmotic swelling test and normal acrosomal reaction, respectively. The highest (P < 0.05) motility, acrosomal integrity and hypo-osmotic swelling response of spermatozoa was achieved by addition of 1.0 and 2.0 mM BHT to semen extender. However, highest (P < 0.05) viability of spermatozoa was achieved by inclusion of 2.0 mM BHT. The higher concentration of BHT (3.0 mM) reduced the motility, acrosomal integrity, viability and hypo-osmotic swelling response of the spermatozoa compared to other concentration used. In conclusion, BHT when added in the semen extender can improve the semen quality of buffalo bulls.     

The influence of cumulus cells during in vitro fertilization of buffalo (Bubalus bubalis) denuded oocytes that have undergone vitrification

The aim of this work was to evaluate whether providing a support of cumulus cells during IVF of buffalo denuded oocytes submitted to vitrification-warming enhances their fertilizing ability. In vitro matured denuded oocytes were vitrified by Cryotop in 20% EG + 20% of DMSO and 0.5 M sucrose and warmed into decreasing concentrations of sucrose (1.25 M-0.3M). Oocytes that survived vitrification were fertilized: 1) in the absence of a somatic support (DOs); 2) in the presence of bovine cumulus cells in suspension (DOs+susp); 3) on a bovine cumulus monolayer (DOs+monol); and 4) with intact bovine COCs in a 1:1 ratio (DOs+COCs). In vitro matured oocytes were fertilized and cultured to the blastocyst stage as a control.An increased cleavage rate was obtained from DOs+COCs (60.9%) compared to DOs, DOs+susp (43.6 and 38.4, respectively; P < 0.01) and DOs+monol (47.5%; P < 0.05). Interestingly, cleavage rate of DOs+COCs was similar to that of fresh control oocytes (67.8%). However, development to blastocysts significantly decreased in all vitrification groups compared to the control (P < 0.01).In conclusion the co-culture with intact COCs during IVF completely restores fertilizing capability of buffalo denuded vitrified oocytes, without improving blastocyst development.     

Improvement of liquid and frozen-thawed semen quality of Nili-Ravi buffalo bulls (Bubalus bubalis) through supplementation of fat

The aim of the study was to investigate the effects of dietary fat on quality of liquid and frozen-thawed semen of Nili-Ravi buffalo bulls. Adult bulls (n = 21) were fed a balanced ration (Con; n = 7) or the same ration either containing sunflower oil (SF-O; n = 7) or whole sunflower seeds (SF-S; n = 7) for 63 days. Body weight and body condition score of each bull was recorded on days 0, 30 and 60 of the experiment. Semen was collected on days 39, 46, 53 and 60, frozen by a fast method and stored at −196 °C for 24 h. Sperm motility was assessed using a bright field microscope. Plasma membrane integrity of fresh and frozen-thawed spermatozoa was assessed using a hypo-osmotic swelling (HOS) assay. The concentration of spermatozoa and volume of semen was not different among groups on various days of collection. Sunflower-enriched diets did not affect the motility and number of HOS-positive spermatozoa in the fresh semen. Motility and HOS of post-thawed spermatozoa were higher (p < 0.05) in bulls fed the sunflower-enriched diets. Similarly, diets did not affect the body condition score and body weight of bulls. In conclusion, feeding of sunflower oil or sunflower seed as fat sources can improve the quality of buffalo bull spermatozoa.     

Improvement in buffalo (Bubalus bubalis) spermatozoa functional parameters and fertility in vitro: Effect of insulin-like growth factor-I

The aim of the current study was to assess the effect of insulin-like growth factor-I (IGF-I; 100 ng/mL) on buffalo (Bubalus Bubalis) sperm functional parameters related to in vitro fertilization. The acrosin activity (the mean diameter of halo formation in micrometers) was significantly higher in the IGF-I group (14.17 ± 1.51) compared with that in the control group (9.50 ± 0.36) at 2 h incubation. The mitochondrial membrane potential (per cent) was significantly higher in the IGF-I group compared with that in the control group at 30 min (33.27 ± 2.62 vs. 26.71 ± 1.02), 60 min (24.24 ± 3.45 vs. 18.77 ± 2.09), and 90 min (22.86 ± 3.02 vs. 16.92 ± 1.24) incubation. The percentage of spermatozoa positive for sperm nuclear chromatin decondensation (NCD) differed significantly between the groups at 90 and 120 min incubation. The comet length was significantly lower in the IGF-I group compared with that in the control group at 2 h incubation. The percentage of fragmented DNA in the tail did not differ significantly between the groups at 2 h incubation. The percentage of acrosomal-reacted spermatozoa did not differ significantly between the IGF-I and the control groups at 4 h (41.12 ± 6.44 vs. 43.53 ± 5.05) incubation. The cleavage rate (per cent) was significantly higher in the IGF-I-treated group (56.73 ± 3.70) compared with that in the control group (44.85 ± 2.15). The current study suggests that the addition of IGF-I prevents deterioration of sperm functional parameters and fertility.     

Influence of season on corpus luteum structure and function and AI outcome in the Italian Mediterranean buffalo (Bubalus bubalis)

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 < 0.05) during the breeding season (58.0%) than the transitional period (45.6%) and this was primarily the result of a lower (P < 0.05) late embryonic mortality during the breeding season (7.3%) compared with the transition period (23%). Circulating concentrations of P4 on Days 10 and 20 after AI were greater (P < 0.01) during the breeding season (4.6 ± 0.3 and 3.4 ± 0.2, respectively) than during the transition period (1.6 ± 0.12 and 1.8 ± 0.2, respectively), and this was independent of reproductive status as there was no interaction between pregnancy and season. Corpus luteum time average medium velocity at Day 10 after AI was greater (P < 0.01) during the breeding season (19.3 ± 1.5) than in the transitional period (8.3 ± 0.7). There were positive correlations in pregnant buffaloes between CL time average medium velocity and P4 concentrations on Day 10 (r = 0.722; P < 0.01) and Day 20 (r = 0.446; P < 0.01) after AI. The findings were interpreted to indicate that relationships between CL vascularization, CL function, and pregnancy outcome in AI in buffaloes are consistent across the breeding season and transition period to the nonbreeding season. The distinction between the breeding season and the transition period is the relatively low proportion of buffaloes that have CL function and P4 concentrations required to establish a pregnancy during the transition period, which is manifested in a greater incidence of embryonic mortality.     

Spermatogonia-specific proteins expressed in prepubertal buffalo (Bubalus bubalis) testis and their utilization for isolation and in vitro cultivation of spermatogonia

Buffalo is an economically important livestock species in Asia. Little is known about male germ line technology owing to lack of sufficient understanding regarding expression of germ- and somatic-cell specific-proteins in the testis. In this study, we identified UCHL-1 (PGP 9.5) and lectin- Dolichos biflorus agglutinin (DBA) as specific markers for spermatogonia in buffalo testis. Expression of germ-cell and pluripotency-specific proteins such as DDX4 (VASA) and POU5F1 (OCT3/4) were also present in spermatogonia. Interestingly, the expression of somatic cell-specific proteins such as VIMENTIN and GATA4 were also detected in germ cells. Using two-step enzymatic digestion followed by differential plating and Percoll density-gradient centrifugation, an approximately 55% spermatogonia-enriched cell population could be obtained from the prepubertal buffalo testis. Isolated spermatogonia could survive and proliferate in vitro in DMEM/F12 medium containing 10% fetal bovine serum in the absence of any specific growth factors for a week. Cultured spermatogonia showed DBA affinity and expressed DDX4 and POU5F1. These results may help to establish a long-term culture system for buffalo spermatogonia.     

Treatment of donor cells with trichostatin A improves in vitro development and reprogramming of buffalo (Bubalus bubalis) nucleus transfer embryos

It has been reported that buffalo (Bubalus bubalis) embryos reconstructed by somatic cell nucleus transfer (SCNT) can develop to the full term of gestation and result in newborn calves. However, the developmental competence of reconstructed embryos is still low. Recently, it has been reported that treating donor cells or embryos with trichostatin A (TSA) can increase the cloning efficiency in some species. Thus, the present study was undertaken to improve the development of buffalo SCNT embryos by treatment of donor cells (buffalo fetal fibroblasts) with TSA and explore the relation between histone acetylation status of donor cells and developmental competence of SCNT embryos. Treatment of donor cells with either 0.15 or 0.3 μM TSA for 48 hours resulted in a significant increase in the cleavage rate and blastocyst yield of SCNT embryos (P < 0.05). Meanwhile, the expression level of HDAC1 in donor cells was also decreased (0.4–0.6 fold, P < 0.05) by TSA treatment, although the expression level of HAT1 was not affected. Further measurement of the epigenetic maker AcH4K8 in buffalo IVF and SCNT embryos at the eight-cell stage revealed that the spatial distribution of acH4K8 staining in SCNT embryos was different from the IVF embryos. Treatment of donor cells with TSA resulted in an increase in the AcH4K8 level of SCNT embryos and similar to fertilized counterparts. These results suggest that treatment of donor cells with TSA can facilitate their nucleus reprogramming by affecting the acetylated status of H4K8 and improving the in vitro development of buffalo SCNT embryos. The AcH4K8 status at the eight-cell stage can be used as an epigenetic marker for predicting the SCNT efficiency in buffalos.     

Impact of buserelin acetate or hCG administration on day 12 post-ovulation on subsequent luteal profile and conception rate in buffalo (Bubalus bubalis)

The present study investigated the impact of gonadotropic hormone administration on day 12 post-ovulation on subsequent luteal profile and conception rate in buffaloes. All the buffaloes (n = 48) were estrus synchronized by a synthetic analogue of prostaglandin F_2α (PGF_2α), administered 11 days apart, followed by insemination during mid to late estrus. To examine the effect of mid-luteal phase hormonal treatment, buffaloes were randomly divided into control (normal saline, n = 14), d12-BA (buserelin acetate, 20 μg, n = 17) and d12-hCG (hCG, 3000 IU, n = 17) groups. Ovaries were scanned on the day of induced estrus to measure the preovulatory follicle (POF) diameter and on days 5, 12, 16 and 21 post-ovulation to examine the alterations in corpus luteum (CL) diameter. On the day of each sonography, blood samples were collected for the estimation of plasma progesterone. In treatment groups, luteal profile (CL diameter and plasma progesterone) on day 16–21 post-ovulation was better (P < 0.05) as well as first service conception rate was higher (52.9% in each treatment group vs. 28.6%, P > 0.05) compared to controls. All the pregnant buffaloes exhibited higher (P < 0.05) plasma progesterone on various post-ovulation days than their respective non-pregnant counterparts. Treatment-induced accessory corpus luteum (ACL) formation was observed in 58.8 per cent and 70.6 per cent buffaloes of d12-BA and d12-hCG group, respectively, that also had higher (P < 0.05) plasma progesterone compared to controls. Compared to the spontaneous CL, the diameter of ACL was less (P < 0.05) in the treatment groups. In conclusion, buserelin acetate and hCG administration on day 12 post-ovulation leads to accessory CL formation, improves luteal profile and consequently increases conception rate in buffaloes.     

Expression and localization of locally produced growth factors regulating lymphangiogenesis during different stages of the estrous cycle in corpus luteum of buffalo (Bubalus bubalis)

Recent experiments using expression, immunolocalization, and cell culture approaches have provided leading insights into regulation of luteal angiogenesis by different growth factor systems and its role in the function of corpus luteum (CL) in buffalo. On the contrary, lymphangiogenesis and its regulation in the CL are still poorly understood. The aim of this study was to evaluate the expression and localization of lymphangiogenic factors (vascular endothelial growth factor [VEGF]-C and VEGFD), their receptor (VEGFR3), and lymphatic endothelial marker (LYVE1) in bubaline CL during different stages of the estrous cycle and to investigate functional role of VEGFC and VEGFD in luteal lymphangeogenesis. The mRNA and protein expression of VEGFC, VEGFD, and VEGFR3 was significantly greater in mid and late luteal phases, which correlated well with the expression of LYVE1. The lymphangiogenic factors were localized in luteal cells, exclusively in the cytoplasm. Immunoreactivity of VEGFC was greater during midluteal phase and that of VEGFD was greater during the mid and late luteal phases. Luteal cells were cultured in vitro and treated for different time duration (24, 48, and 72 hours) with VEGFC and VEGFD each at 50, 100, and 150 ng/mL concentration and VEGFC with VEGFD at 100 ng/mL concentration. The temporal increase in LYVE1 mRNA expression was significant (P < 0.05) in VEGFC and VEGFC with VEGFD treatment and no significant change was seen in VEGFD treatment. Thus, it seems likely that VEGFD itself has little role in lymphangiogenesis but along with VEGFC it might have a synergistic effect on VEGFR3 receptors for inducing lymphangiogenesis. In summary, the present study provided evidence that VEGFC and VEGFD, and their receptor VEGFR3, are expressed in bubaline CL and are localized exclusively in the cell cytoplasm, suggesting that these factors have a functional role in lymphangiogenesis of CL in buffalo.     

Comparison of endocrine changes,timing of ovulations,ovarian follicular growth,and efficacy associated with Estradoublesynch and Heatsynch protocols in Murrah buffalo cows (Bubalus bubalis)

Poor estrus expression and the difficulty encountered in predicting the time of ovulation compromise the reproductive efficiency of Murrah buffalo cows. Synchronization of ovulation and timed artificial insemination are able to precisely control the time of ovulation and thus avoid the need for estrus detection. Recently, the Estradoublesynch protocol (administration of a PGF2α injection 2 days before Heatsynch protocol; GnRH 0, PGF2α 7, estradiol benzoate [EB] 8) was developed that precisely synchronized ovulation twice, i.e., after GnRH and EB injections and resulted in satisfactory pregnancy rates in Murrah buffaloes. The present study was conducted on 104 cycling and 31 anestrus buffaloes to compare (1) the endocrine changes, timing of ovulations, ovarian follicular growth, and efficacy of Estradoublesynch and Heatsynch protocols in cycling and (2) the efficacy of Estradoublesynch and Heatsynch protocols for the improvement of fertility in cycling and anestrus Murrah buffalo cows. Ovulation was confirmed after all GnRH and EB treatments by ultrasonographic examination at 2-hour intervals. Plasma progesterone and total estrogen concentrations were determined in blood samples collected at daily intervals, beginning 2 days before the onset of protocols until the day of second ovulation detection. Ovulatory follicle size was measured by ultrasonography at six time points (first PGF2α administration of Estradoublesynch protocol every 2 days before the onset of Heatsynch protocol, GnRH administration of both protocols, 2 hours before ovulation detection after GnRH administration of both protocols, second PGF2α injection of Estradoublesynch protocol, PGF2α injection of Heatsynch protocol, EB injection of both protocols and, 2 hours before ovulation detection after EB administration of both protocols). Plasma LH, total estrogen, and progesterone concentrations were determined in blood samples collected at 30-minute intervals for 8 hours, beginning GnRH and EB injections, and thereafter at 2-hour intervals until 2 hours after the detection of ovulation. The first ovulatory rate was significantly higher (P < 0.05) in the Estradoublesynch protocol (84.6%) than that in the Heatsynch protocol (36.4%). The first LH peak concentration (74.6 ± 10.4 ng/mL) in the Estradoublesynch protocol was significantly higher (P < 0.05) than that of the Heatsynch protocol (55.3 ± 7.4 ng/mL). In Estradoublesynch protocol, the total estrogen concentration gradually increased from the day of GnRH administration coinciding with LH peak, and then gradually declined to the basal level until the time of ovulation detection. However, in Heatsynch protocol, the gradual increase in total estrogen concentration after GnRH administration was observed only in those buffalo cows, which responded to treatment with ovulation. In both Estradoublesynch and Heatsynch protocols, ovulatory follicle size increased by treatment with GnRH and EB until the detection of ovulation. The pregnancy rate after the Estradoublesynch protocol (60.0%) was significantly higher (P < 0.05) than that achieved after the Heatsynch protocol (32.5%). Satisfactory success rate using the Estradoublesynch protocol was attributed to the higher release of LH after treatment with GnRH, leading to ovulation in most of the animals and hence creating the optimum follicular size at EB injection for ovulation and pregnancy to occur.