Developmental failure of hybrid embryos generated by in vitro fertilization of water buffalo (Bubalus bubalis) oocyte with bovine spermatozoa

The developmental potential of inter-species hybrid embryos produced by in vitro fertilization of in vitro matured buffalo oocytes with bovine spermatozoa was studied with a view to investigate pre-implantation embryo development and its gross morphology, early embryonic gene expression, and embryonic genome activation. Fertilization events with both buffalo and cattle spermatozoa were almost similar. Overall fertilization rate with cattle spermatozoa was 78.4% was not significantly different from that of buffalo spermatozoa (80.2%). Initial cleavage rate between buffalo and hybrid embryo was also similar, and there was no significant difference in their developmental rate till 8-cell stage (26.0 +/- 4.1 vs. 24.3 +/- 4.8). However, only 5.3% of hybrid embryos developed into blastocyst stage compared to 21.7% in buffalo. mRNA phenotyping of insulin-like growth factor family (Insulin, insulin receptor, IGF-I, IGF-I receptor, IGF-II, and IGF-II receptor) and glucose transporter isoforms (GLUT-I, II, III, IV) in hybrid embryos clearly showed that these molecules were not expressed after 8-cell stage onward. Similarly, as observed in buffalo embryos, incorporation of (35)S-methionine and (3)H-uridine could not be observed in hybrid embryos from 8-cell stage onward. This suggests that the maternal-zygotic genome activation did not occur in hybrid embryos. Differential staining also showed that the blastomere stopped dividing after 8-cell stage. Collectively, these parameters clearly showed that there was developmental failure of hybrid embryos.     

A set of polymorphic DNA microsatellites useful in swamp and river buffalo (Bubalus bubalis)

DNA microsatellites have found widespread application in gene mapping, pedigree determination and population genetics. In closely related species such as bovids, heterologous polymerase chain reaction (PCR) primers may in some cases be used, bypassing the need to isolate and characterize microsatellite-containing sequences and design PCR primers. We report on the ability of a set of eighty bovine derived DNA microsatellite primers to amplify sequences in the two types (swamp and river) of water buffalo ( Bubalus bubalis ). Number of alleles and per cent heterozygosities in a large number of animals were determined on a subset of microsatellite loci selected on the robustness of the primers. These loci will form the basis of a set of polymorphic DNA markers for use in water buffalo.     

Effect of treatment with recombinant bovine somatotropin on responses to superovulatory treatment in swamp buffalo (Bubalus bubalis)

The objective of this study was to determine the effect of treatment with recombinant bovine somatotropin (rBST) on the response to superovulatory treatment in swamp buffalo. Estrous cycles of 16 buffalo cows were synchronized by intravaginal administration of progesterone and estradiol benzoate, and the cows were then randomly divided into 2 groups. The rBST-treated group received 250 mg of a sustained-release formula of rBST on Day 4 after progesterone implantation, whereas the control group did not receive rBST. Both groups were then given a superovulatory regimen of twice daily injections of FSH for 3.5 d (total dose of 260 mg, i.m.), between Days 9 and 11 after administration of progesterone. The cows were bred naturally 1 d after the last FSH injection, then 6 d after breeding they were slaughtered, and their reproductive tracts were removed. The numbers of corpora lutea (CL) and follicles were recorded, and embryos were flushed out of the uterine horns. There were no significant differences between the rBST-treated and control cows for the mean numbers (+/- SEM) of CL (6.0 +/- 2.2 vs 4.3 +/- 1.1), follicles (15.9 +/- 4.1 vs 19.8 +/- 2.9), or total embryos recovered per collection (4.5 +/- 1.6 vs 2.3 +/- 1.0). However, there were significant differences between rBST-treated and control cows for the numbers of transferable embryos per collection (3.0 +/- 1.0 vs 0.8 +/- 0.3; P < or = 0.05) and the overall proportion of transferable embryos (75 vs 33%; P < or = 0.01). The results of this study show that pretreatment of swamp buffalo with rBST significantly increases the production of transferable embryos in response to superovulation.     

Genetic differentiation of water buffalo (Bubalus bubalis) populations in China, Nepal and south-east Asia: inferences on the region of domestication of the swamp buffalo

Data from three published studies of genetic variation at 18 microsatellite loci in water buffalo populations in China (18 swamp type, two river type), Nepal (one wild, one domestic river, one hybrid) and south-east Asia (eight swamp, three river) were combined so as to gain a broader understanding of genetic relationships among the populations and their demographic history. Mean numbers of alleles and expected heterozygosities were significantly different among populations. Estimates of θ (a measure of population differentiation) were significant among the swamp populations for all loci and among the river populations for most loci. Differentiation among the Chinese swamp populations (which was due primarily to just one population) was much less than among the south-east Asian. The Nepal wild animals, phenotypically swamp type but genetically like river type, are significantly different from all the domestic river populations and presumably represent the ancestral Bubalus arnee (possibly with some river-type introgression). Relationships among the swamp populations (D(A) genetic distances, principal component analysis and structure analyses) show the south-east Asian populations separated into two groups by the Chinese populations. Given these relationships and the patterns of genetic variability, we postulate that the swamp buffalo was domesticated in the region of the far south of China, northern Thailand and Indochina. Following domestication, it spread south through peninsular Malaysia to Sumatra, Java and Sulawesi, and north through China, and then to Taiwan, the Philippines and Borneo.     

Optimization of cryopreservation of buffalo (Bubalus bubalis) blastocysts produced by in vitro fertilization and somatic cell nuclear transfer

The objective of this study was to optimize cryopreservation conditions for buffalo in vitro produced (IVP) embryos. The in vitro fertilized (IVF) and somatic cell nuclear transferred (SCNT) blastocysts were vitrified with either 40% ethylene glycol (EG), 25% EG + 25% dimethylsulfoxide (DMSO), or 20% EG + 20% DMSO + 0.5 m sucrose, and the IVF blastocysts produced from abattoir-derived ovaries were also slow-frozen with either 10% EG or 0.05 m trehalose dehydrate + 1.8% EG + 0.4% BSA. Cryosurvival rates of blastocysts harvested on various days or at various developmental stages were also examined. In this study: (1) vitrification with 20% EG + 20% DMSO + 0.5 m sucrose had the best cryopreservation efficiency; (2) IVF and SCNT blastocysts had similar cryotolerance (P > 0.05); (3) after thawing, slow-frozen blastocysts reexpanded earlier than the vitrified blastocysts (P < 0.01); (4) cryosurvival rate of expanded blastocysts was higher than that of early blastocysts (P < 0.05); (5) cryosurvival rates of Days 5 to 7 blastocysts (Day 0 = day of IVF or SCNT) were higher than those of Days 8 to 9 blastocysts (P < 0.01); and (6) after embryo transfer, pregnancy rates for fresh and cryopreserved blastocysts were not different (P > 0.05). In conclusion, vitrification of Days 6 to 7 expanded blastocysts with 20% EG + 20% DMSO + 0.5 m sucrose was optimal for cryopreservation of buffalo IVP embryos.     

Mannose-binding lectin haplotypes influence Brucella abortus infection in the water buffalo (Bubalus bubalis)

A case-control study established that the haplotype pair HYA/HYA at the MBL (mannose binding lectin) locus of water buffalo is associated with resistance to Brucella abortus infection (P < 10⁻⁷) and the haplotype pairs LYD/LYD with susceptibility to the same pathogen (P < 10⁻⁷). The subjects included in the present study were tested twice—at a 1-month interval—for the presence of anti-B. abortus antibodies in the serum by agglutination, complement fixation and flow cytometry. Cases (335 subjects) included animals consistently positive to all these tests; controls (335 subjects) comprised animals exposed yet negative by the same tests. The serum from genetically resistant subjects displayed in vitro significantly higher antibacterial activity compared to the serum from genetically susceptible subjects, lending biological significance to the results from the association study. Inhibition of the antibacterial activity following heat treatment of the serum, addition of specific MBL inhibitors (EDTA, mannose, N-acetyl-d-glucosamine) or anti-human MBL antiserum provide convincing evidence that the antibacterial activity present in the serum results from the interaction between MBL and B. abortus. A replication study (comprising 100 cases and 100 controls) confirmed the results from the original study.     

Influence of sire breed (Bos indicus versus Bos taurus) and interval from slaughter to oocyte aspiration on heat stress tolerance of in vitro-produced bovine embryos

Based on in vitro experiments, Bos indicus embryos were more resistant to heat stress (HS) than Bos taurus embryos. To increase knowledge regarding differences between Bos indicus and Bos taurus in resistance to HS, the primary objective of this study was to determine if tolerance to HS is due to the breed, origin of the oocyte, sperm, or both. Additionally, the influence of the interval between ovary acquisition (in the abattoir) and oocyte aspiration in the laboratory, on early embryo development was ascertained. Oocytes were collected from Nelore and Holstein cows in an abattoir; 4.0 or 6.5 h later, oocytes were aspired in the laboratory, and then matured and fertilized using semen from Nelore (N), Gir (GIR), or Holstein (H) bulls. Ninety-six h post insemination (hpi), embryos with ≥ 16 cells were divided in two groups: control and HS. In the control group, embryos were cultured at 39°C, whereas in the HS group, embryos were subjected to 41°C for 12 h, and then returned to 39°C. Rates of cleavage, and formation of morula and blastocysts were higher (P < 0.05) for oocytes aspirated at 4.0 versus 6.5 h after ovaries were acquired. Heat stress decreased rates of blastocyst formation for all breeds (N × N; H × H; and H × GIR) and in both time intervals (4.0 and 6.5 h). However, N × N had higher cleavage rate (P < 0.05) in both time intervals when compared with H × H and H × GIR. In addition, Nelore oocytes fertilized with Nelore semen (N × N) had higher blastocyst yields (P < 0.05) in the control and HS group, when compared with the other two breeds (H × H and H × GIR). We concluded that the breed of origin of the oocyte was more important than that of the sperm for development of thermotolerance, because bull breed did not influence embryo development after HS, and in vitro early embryonic development was impaired by increasing (from 4 to 6.5 h) the interval between ovary acquisition and oocyte aspiration.     

Relationship of scrotal circumference and testicular volume to age and body weight in the swamp buffalo (Bubalus bubalis)

Scrotal circumference (SC) and testicular volume (TV) were measured in 65 swamp buffalo bulls ranging in age from 7 to 60 months and weighing 130 kg to 560 kg. Ages and body weight (BW) were recorded for each male at the time of measurement to find out if they correlated with SC and TV. SC and TV increased linearly and correlated significantly with age and BW (SC vs age: r = 0.74, p<0.01; SC vs BW: r = 0.88, p<0.01; TV vs BW: r = 0.82, p<0.01). SC measurements ranged from 15.1 +/- 1.1 cm to 24.0 +/- 0.4 cm for ages ranging from 10.0 +/- 0.6 to 48.5 +/- 6.3 months, revealing that testicular size in swamp buffaloes was very much smaller than domestic cattle. The SC norms distributed with age would be useful in the evaluation of swamp buffalo males for breeding soundness.     

Pregnancy rates following AI with sexed semen in Mediterranean Italian buffalo heifers (Bubalus bubalis)

The use of sexed semen in farm animal production and genetic improvement has been shown to be feasible with variable degree of efficiency in a number of species, and proved to be economically viable in cattle. In the last two decades, various newly developed reproductive technologies applicable in buffaloes have mushroomed. Recently, following the birth of the first buffalo calves using AI with sexed semen, commercial interest to exploit sexing of semen in this species too is aroused. In order to verify the successful adoption of this technology in the buffalo, the present study on the use of sexed semen for AI was carried out and compared with conventional artificial insemination using nonsexed semen. A total of 379 buffalo heifers were used for synchronization of ovulation using the Presynch protocol in the South of Italy. Selected animals at the time of AI were randomly allocated to three different experiment groups: (1) 102 animals subjected to AI in the body of the uterus with sexed semen (SS body); (2) 104 animals subjected to AI in the horn of the uterus with sexed semen (SS horn); and (3) 106 animals subjected to AI in the body of the uterus with conventional nonsexed semen (NSS body). Semen of three buffalo bulls was sexed by a collaborating company and commercially distributed in 0.25 mL straws with a total of 2 million sexed spermatozoa. Pregnancy rates were first assessed at Day 28 following AI, and rechecked at Day 45 by ultrasound. Pregnancy rates were nonsignificantly different between animals inseminated with sexed or nonsexed semen: 80/206 (38.8%) and 40/106 (37.7%), respectively (P = 0.85). However, site of insemination of sexed semen affected pregnancy rate significantly as higher pregnancy rates were obtained when sexed semen was deposited into the body rather than the horn of the uterus: 46/101 (45.5%) and 34/105 (32.3%), respectively (P = 0.05). In conclusion, the use of sexed semen in buffalo heifers gave satisfactory and similar pregnancy rates when compared with conventional nonsexed semen. Deposition of sexed semen into the body of the uterus, however, increased pregnancy rates significantly.     

Comparison of non-linear models to describe the lactation curves for milk yield and composition in buffaloes (Bubalus bubalis)

In order to describe the lactation curves of milk yield (MY) and composition in buffaloes, seven non-linear mathematical equations (Wood, Dhanoa, Sikka, Nelder, Brody, Dijkstra and Rook) were used. Data were 116 117 test-day records for MY, fat (FP) and protein (PP) percentages of milk from the first three lactations of buffaloes which were collected from 893 herds in the period from 1992 to 2012 by the Animal Breeding Center of Iran. Each model was fitted to monthly production records of dairy buffaloes using the NLIN and MODEL procedures in SAS and the parameters were estimated. The models were tested for goodness of fit using adjusted coefficient of determination root means square error (RMSE), Durbin–Watson statistic and Akaike’s information criterion (AIC). The Dijkstra model provided the best fit of MY and PP of milk for the first three parities of buffaloes due to the lower values of RMSE and AIC than other models. For the first-parity buffaloes, Sikka and Brody models provided the best fit of FP, but for the second- and third-parity buffaloes, Sikka model and Brody equation provided the best fit of lactation curve for FP, respectively. The results of this study showed that the Wood and Dhanoa equations were able to estimate the time to the peak MY more accurately than the other equations. In addition, Nelder and Dijkstra equations were able to estimate the peak time at second and third parities more accurately than other equations, respectively. Brody function provided more accurate predictions of peak MY over the first three parities of buffaloes. There was generally a positive relationship between 305-day MY and persistency measures and also between peak yield and 305-day MY, calculated by different models, within each lactation in the current study. Overall, evaluation of the different equations used in the current study indicated the potential of the non-linear models for fitting monthly productive records of buffaloes.     

Evaluation of sperm functional attributes in relation to in vitro sperm-zona pellucida binding ability and cleavage rate in assessing frozen thawed buffalo (Bubalus bubalis) semen quality

The objective of this study was to evaluate sperm functional attributes in relation to in vitro sperm-zona binding ability and cleavage rate in assessing frozen thawed buffalo (Bubalus bubalis) semen quality. Frozen-thawed forty-eight ejaculates from eight Surti buffalo bulls (six ejaculates/bull) obtained by artificial vagina were used. Frozen semen from each bull was thawed, pooled, and subjected for sperm functional (six replicates) and in vitro fertilization (four replicates) tests. The progressive forward motility, plasmalemma functional integrity assessed by fluorogenic [6-carboxyfluorescein diacetate (CFDA), and propidium iodide (PI)], hypoosmotic swelling (HOS), and hypoosmotic swelling-Giemsa (HOS-G) test, mitochondrial membrane potential, sperm nuclear morphology, the number of sperm bound to zona and cleavage rate differed significantly (P < 0.05) between bulls. When the animals were grouped based on cleavage rate (group I, >40% cleavage rate, n = 5, and group II, <40% cleavage rate, n = 3), in vitro fertility parameters and all the sperm functional attributes except sperm nuclear morphology differed significantly (P < 0.05). The proportions of sperm with functional plasmalemma in the tail and intact acrosome assessed by HOS-G test (25.33, range: 17.48–40.27) were significantly (P < 0.001) lower than the functional plasmalemma in the tail assessed by HOS test (39.80, range: 27.85–54.67). The number of sperm bound to zona had significant correlations with the mitochondrial membrane potential (r = 0.90, P < 0.01) and plasmalemma integrity (fluorogenic, r = 0.74 and HOS, r = 0.79, P < 0.05) and HOS-G, r = 0.87, P < 0.01). The cleavage rate had significant (P < 0.05) correlations with the mitochondrial membrane potential (r = 0.70) and plasmalemma integrity measured by HOS-G test (r = 0.68). The present study indicates that these attributes could represent important determinants of buffalo sperm quality influencing cleavage rate.     

Effects of two estradiol esters (benzoate and cypionate) on the induction of synchronized ovulations in Bos indicus cows submitted to a timed artificial insemination protocol

The effects of estradiol benzoate (EB) and estradiol cypionate (EC) on induction of ovulation after a synchronized LH surge and on fertility of Bos indicus females submitted to timed AI (TAI) were evaluated. In Experiment 1, ovariectomized Nelore heifers were used to evaluate the effect of EB (n = 5) and EC (n = 5) on the circulating LH profile. The LH surge timing (19.6 and 50.5 h; P = 0.001), magnitude (20.5 and 9.4 ng/mL; P = 0.005), duration (8.6 and 16.5 h; P = 0.001), and area under the LH curve (158.6 and 339.4 ng/mL; P = 0.01) differed between the EB and EC treatments, respectively. In Experiment 2 (follicular responses; n = 60) and 3 (pregnancy per AI; P/AI; n = 953) suckled Bos indicus beef cows submitted to an estradiol/progesterone-based synchronization protocol were assigned to receive one of two treatments to induce synchronized ovulation: 1 mg of EB im 24 h after progesterone (P4) device removal or 1 mg of EC im at P4 device removal. There was no difference (P > 0.05) between EB and EC treatments on follicular responses (maximum diameter of the ovulatory follicle, 13.1 vs. 13.9 mm; interval from progesterone device removal to ovulation, 70.2 vs. 68.5 h; and ovulation rate, 77.8 vs. 82.8%, respectively). In addition, P/AI was similar (P < 0.22) between the cows treated with EB (57.5%; 277/482) and EC (61.8%; 291/471). In conclusion, despite pharmacologic differences, both esters of estradiol administered either at P4 device removal (EC) or 24 h later (EB) were effective in inducing an LH surge which resulted in synchronized ovulations and similar P/AI in suckled Bos indicus beef cows submitted to TAI.