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SummarySomatic cell nuclear transfer (NT) is associated with aberrant changes in epigenetic reprogramming that impede the development of embryos, particularly during zygotic genome activation. Here, we characterized epigenetic patterns of H3K4me3, H3K9me3, and H3K27me3 in mouse NT embryos up to the second cell cycle (i.e. four-celled stage) during zygotic genome activation. In vivo fertilized and parthenogenetically activated (PA) embryos served as controls. In fertilized embryos, maternal and paternal pronuclei exhibited asymmetric H3K4me3, H3K9me3, and H3K27me3 modifications, with the paternal pronucleus showing delayed epigenetic modifications. Higher levels of H3K4me3 and H3K9me3 were observed in NT and PA embryos than in fertilized embryos. However, NT embryos exhibited a lower level of H3K27me3 than PA and fertilized embryos from pronuclear stage 3 to the four-celled stage. Our finding that NT embryos exhibited aberrant H3K4me3, H3K9me3, and H3K27me3 modifications in comparison with fertilized embryos during early zygotic genome activation help to unravel the epigenetic mechanisms of methylation changes in early NT reprogramming and provide an insight into the role of histone H3 in the regulation of cell plasticity during natural reproduction and somatic cell NT.

We injected mouse zygotes with combinations of Cas9 protein, Cas9 mRNA, and two gRNAs targeting a single exon of type I interferon receptor (IFNAR1) to determine the gene targeting efficiencies. Cas9 protein produced on-target mutations more efficiently than Cas9 mRNA when each was used with a single gRNA, regardless of which gRNA was used. When Cas9 mRNA and Cas9 protein were co-injected, the on-target efficiency could reach 97.0% when both gRNAs were used, which was higher than when either gRNA was used alone (61.3% and 75.5%, respectively; P

Blastocysts hatch from the zona pellucida (ZP) to enable implantation into the uterine endometrial epithelium, but little is known regarding the effect of hatching sites on pregnancy outcomes. Murine hatching embryos were categorized into five groups based on initial trophectoderm projection (TEP)/ZP position corresponding to the inner cell mass center. In blastocysts (3.5 dpc) post-12 hours in vitro culture, TEP rates of A-site (44.4%) and B-site (38.6%) embryos were higher than those of C-site (12.5%) and D-site (3.1%) embryos, while the O-site (1.4%) was the lowest (P < .05). Post-ET A-site (55.6%) and B-site (65.6%) birth rates were higher than those of C-site embryos (21.3%) and controls (P < .05). Furthermore, live birth rate of B-site embryos remained higher than C-site embryos (68.8% vs 31.3%; P < .05) when both were transferred into the same recipients. Different TEP site blastocysts exhibited different implantation competences: the implantation rate of C-site embryos was lower than that of both A- and B-site groups (67.7% vs 84.3% and 83.2%, respectively; P < .05) at 2 days post-ET. C-site embryos also had a distinctly higher ratio of developmental defects (47.5%) than A- and B-site embryos (22.5% and 14.6%, respectively), with implantation failure mainly associated with poor birth rate, a finding corroborated by differential gene expression analysis such as LIF, LIFR, and S100a9. Surprisingly, acidified Tyrode's solution (AAH)-treated B-site blastocysts had a significantly increased birth rate (77.1%) than C-site (55.3%) and controls (43.4%). Site specificity and differential gene expression during embryo hatching can be applied in ART screening. More importantly, assisted hatching by AAH is effective and feasible for improving pregnancy and term development, particularly at the B-site, for humans and in animal husbandry.

The domestic buffalo (Bubalus bubalis) is characterized by a worldwide distribution and its production is expressed within both intensive systems and marginal areas. Although a domesticated species since long time, over the years, the buffalo has scarcely benefitted of the strategies held in place for genetic improvement, as opposed to the cattle counterpart. Only at the beginning of the 1990s, the problems related to the enhancement of buffalo productivity through the implementation of reproductive technologies, namely the use of AI, have begun to be tackled. In this chapter, a report on the use of reproductive technologies in buffaloes will be presented, highlighting the early attempts at such and the remarkable more recent achievements in different areas, ranging from in vivo and in vitro embryo production to transgenesis, cloning, and stem cell technologies. Finally, an outlook to more pragmatic aspects will be given, such as the cost-to-benefit ratio of some of the reproductive strategies discussed.

Goat oocyte in vitro maturation is associated with a variable efficiency of embryo development after in vitro fertilization (IVF). Here, we developed a novel maturation procedure to evaluate the cellular effect of cysteamine (Cys), leukemia inhibitory factor (LIF) and Y27632 on oocyte in vitro maturation in native Chinese Yangtze river white goats. Oocytes were collected by slicing ovary tissues and matured for 24 h in vitro prior to IVF. Presumptive fertilized oocytes were cultured in embryo media for 8 days. Maturation rates were similar in gonadotropin basal maturation medium and the same medium supplemented with Cys, LIF, or Y27362 (41.0-48.0%; P 0.05). However, when two substances were co-supplemented into the medium, the maturation rate was higher in the Cys+LIF group than in the LIF+Y27362 and Cys+Y27362 groups (60.0% vs. 43.1% and 25.8%, respectively; P 0.05). Co-supplementation of all three substances into the medium achieved the highest maturation rate (67.5%; P 0.05). Compared with oocytes in gonadotropin basal maturation medium, those in medium supplemented with Cys showed increased fertilization (56.1% vs. 72.1%), cleavage (36.7% vs. 44.8%), and blastocyst development (1.7% vs. 4.2%), respectively (P 0.05). Cys+LIF supplementation further improved fertilization (81.6%), cleavage (54.9%), and blastocyst development (6%; P 0.05). Furthermore, combined supplementation of all three substances resulted in the best fertilization (84.9%), cleavage (70.7%), and blastocyst development (10.3%; P 0.05). Resultant IVF blastocysts possessed an average cell number as high as 276 ± 45 per embryo. This is the first study to report increased efficiency of caprine oocyte maturation by combined Cys, LIF, and Y27632 supplementation into basal maturation medium, leading to improved fertilization and embryo development in vitro post-IVF.

The combined effect of six consecutive timed artificial inseminations (TAIs) on pregnancy rates, following two different synchronization protocols on buffalo heifers, over a period of seven months typically characterized by low breeding performances, were investigated in this study. A total of 2189 TAIs were performed on 1463 buffalo heifers within a large buffalo farm in the south of Italy. Individual animals were allowed to undergo synchronization protocol (either a slightly modified Ovsynch or Progesterone treatment) and TAI until establishment of pregnancy or else for not more than six consecutive times. Semen of seven proven bulls was used throughout the study, which was carried out from March to September of the same year. Therefore, other than the effect given by consecutive TAIs over time, a monthly and a seasonal effect could also be tested, once the entire period was split into a Low Breeding Season (LBS) from March to June, and a Transition to Breeding Season (TBS) from July to September. From the data recorded in this study and the statistical analysis performed, it can be stated that the two protocols for the synchronization of ovulation were similar in efficiency in determining pregnancies with an overall fertility rate of 89.4% when the comparison was run both on a monthly basis or when months were grouped into two different seasons. In addition, an average of 1.83 AI/pregnancy was reported, slightly higher for the Ovsynch when compared to the Progesterone protocol: 1.91 vs 1.70, respectively. Finally, when considering the number of progressive synchronization treatments implemented over time as covariate, neither Ovsynch nor Progesterone treatment significantly affected pregnancy rates following the first of the six synchronization sessions. However, repeating the synchronization procedure, the progesterone based protocol resulted in significantly higher probability of success in terms of established pregnancies during the second and third re-synchronization sessions.

The aim of this study was to assess modified droplet vitrification (MDV) for the cryopreservation of early developmental mouse embryos. Mouse embryos were equilibrated in holding solution for 3 min followed by immersion in vitrification solution for 30-45 s, and then three embryos per 3-μL vitrification droplet were directly dropped into liquid nitrogen. Vitrified embryos were warmed to examine their developmental potential both in vitro and in vivo. The results demonstrated that MDV vitrified and warmed embryos had a survival rate of 98.1-99.6% (P0.05); however, blastocyst development post warming and culture in vitro demonstrated that vitrified 4-celled, 8-celled, 16-celled, morulae, and blastocyst embryos had significant higher developmental potentials (94.7-99.5%) than those from zygotes (9.2%) and 2-celled embryos (85.7%) (P0.05). Compared to CryoLoop and CryoTech vitrification, MDV showed similar results with regards to rates of survival, blastocyst development, but with the higher hatching rate (76.1% vs. 64.0-67.3%) (P0.05). Cryopreservation by MDV resulted in a similar blastocyst developmental potential in 4-celled and 16 celled embryos from ICR (94.7-99.5%), C57BL/6J (94.7-96.4%), and their crossbred F1 strain (97.9-98.9%) (P0.05). After embryo transfer of vitrified ICR embryos from 4-celled, 16-celled, morulae and blastocyst stage, 40.7-43.7% of the embryos developed into live offspring (P0.05), but MDV vitrification resulted in the highest birth rate (43.8%) compared to CryoLoop (38.3%) and CryoTech (35.4%) (P0.05), when 4-celled mouse embryos were used for vitrification. Our study clearly demonstrated that MDV is the most efficient vitrification to cryopreserve embryos at least 4-celled and advanced stages, which can be used to preserve important mouse genomes from different strains and different developmental stages.

Growth, weight at birth and daily weight gain (DWG) on 12 water buffalo calves, starting from 6 days of age until completion of weaning, was investigated in this study. Different feeding regimens were given to two groups of animals with regard to daily milk replacer: (1) group 1 (G1) received a double concentration in single administration; whereas (2) group 2 (G2) received the same amount of milk replacer split twice daily. Blood samples were collected from each calf on days 6, 30, 60 and 90 to evaluate acute phase proteins (haptoglobin), bactericide activity, lysozime, total protein content and biochemical parameters. No differences were observed between the two groups in terms of dry matter intake, feed efficiency and live body weight at the end of the study. Interestingly, a significantly (P

The use of sexed semen technology in buffaloes is nowadays becoming more and more accepted by farmers, to overcome the burden of unwanted male calves with related costs and to more efficiently improve production and genetic gain. The aim of this study was to verify the coupling of some variables on the efficiency of pregnancy outcome after deposition of sexed semen through AI. Pluriparous buffaloes from two different farms (N = 152) were screened, selected, and subjected to Ovsynch protocol for AI using nonsexed and sexed semen from four tested bulls. AI was performed in two distinct periods of the year: September to October and January to February. Neither farms nor bulls had a significant effect on pregnancy rates pooled from the two periods. The process for sexing sperm cells did not affect pregnancy rates at 28 days after AI, for nonsexed and sexed semen, respectively 44/73 (60.2%) and 50/79 (63.2%), P = 0.70, and at 45 days after AI, for nonsexed and sexed semen, respectively 33/73 (45.2%) and 33/79 (49.3%), P = 0.60. Pregnancy rate at 28 days after AI during the transitional period of January to February was higher when compared with September to October, respectively 47/67 (70.1%) versus 47/85 (55.2%), P = 0.06. When the same pregnant animals were checked at Day 45 after AI, the difference disappeared between the two periods, because of a higher embryonic mortality, respectively 32/67 (47.7%) versus 40/85 (47.0%), P = 0.93. Hematic progesterone concentration at Day 10 after AI did not distinguish animals pregnant at Day 28 that would or would not maintain pregnancy until Day 45 (P = 0.21). On the contrary, when blood samples were taken at Day 20 after AI, the difference in progesterone concentration between pregnant animals that would maintain their pregnancy until Day 45 was significant for both pooled (P = 0.00) and nonsexed (P = 0.00) and sexed semen (P = 0.09). A similar trend was reported when blood samples were taken at Day 25, being highly significant for pooled, nonsexed, and sexed semen (P = 0.00). Hematic progesterone concentration between the two periods of the year was highly significant for pregnant animals at 28 days from AI when blood samples were taken at Day 20 after AI for pooled, nonsexed, and sexed semen, respectively P = 0.00, 0.00, and 0.06, and for pregnant animals at Day 45 for pooled, nonsexed, and sexed semen, respectively P = 0.00, 0.00, and 0.01. From these results, it can be stated that hematic progesterone concentration measurement since Day 20 after AI can be predictive of possible pregnancy maintenance until Day 45. Furthermore, the transitional period of January to February, although characterized by a higher pregnancy outcome when compared with September to October, suffers from a higher late embryonic mortality as evidenced by a significant different hematic progesterone concentration between the two periods at Day 20 after AI.

We investigated the effects of 5′-end truncated CRISPR RNA-guided Cas9 nuclease (tru-RGN, 17/18 nucleotides) on genome editing capability in NIH/3T3 cells, and its efficiencies on generating Factor VII (FVII) gene-knockout (KO) mice. In cultured cells, RGNs on-target editing activity had been varied when gRNAs was truncated, higher at Site Two (tF7–2 vs. F7–2, 49.5 vs. 30.1%) while lower in other two sites (Site One, tF7–1 vs.F7–1, 12.1 vs. 23.6%; Site Three, tF7–3 vs.F7–3, 7.7 vs 10.9%) (P FVII KO mice were generated with higher efficiency at Site Two (80.1 vs. 35.8%) and Site One (55.0 vs 3.7%) (P 0.05) when compared with standard RGN controls. Knockout FVII mice demonstrated a delayed prothrombin time and decreased plasma FVII expression. Our study first demonstrates that truncated gRNAs to 18 complementary nucleotides and Cas9 nucleases, can effectively generate FVII gene KO mice with a significantly higher efficiency in a site-dependent manner. In addition, the off-target frequency was much lower in KO mice than in cell lines via RGN expression vector-mediated genome editing.

The aim of this study was to investigate the efficiency of in vitro embryo production in cattle utilizing sexed sperm from two bulls and oocytes recovered by OPU. Twenty donor animals were employed in eight OPU replicates: the first four OPU trials were conducted on animals without hormone treatment, and the last four were run on the same animals, following FSH subcutaneous and intramuscular administration. A higher rate of blastocyst development was recorded in stimulated, as compared to nonstimulated animals, (25.2% versus 12.8%, 𝑃 = . 0 0 1 ). Ocytes derived from slaughterhouse (SH) ovaries were also fertilized with sperm from the same bulls. Overall, non-sexed sperm used with oocytes derived from SH ovaries was significantly more efficient for blastocyst development than was sexed sperm with these same SH derived oocytes and sexed sperm with stimulated donor oocytes (39.8% versus 25.0% and 25.2%, 𝑃 = . 0 0 1 ). In conclusion, the use of sexed sperm with OPU-derived oocytes resulted in a significantly higher blastocyst development when donors were hormonally stimulated; furthermore, the level of efficiency achieved was comparable to that attained when the same sexed sperm was tested on oocytes derived from SH ovaries.

The average number of oocytes collected by ovum pick up (OPU) from Bos taurus cattle is8 per live donor. The objective was to determine whether development of small numbers of cattle embryos (produced by OPU and IVF), was enhanced by including "helper" embryos, produced from abbatoir-derived oocytes and embedded in agarose. Oocytes were from abbatoir-derived ovaries (Experiments 1 and 2) or OPU of elite donors (Experiment 3). In Experiment 1, cleaved embryos (2-8 cells), were randomly allocated. Controls were groups of 1, 3, 5, 10, and 20 cleaved embryos cultured in 50 μL serum-free SOF, whereas treatments were groups of 1, 3, and 5 embryos freely cultured along with helpers in groups of either 9, 7 or 5 embedded in agarose per droplet. Therefore, there were 10 cleaved embryos per droplet in combinations of 1 + 9, 3 + 7 or 5 + 5 (free + helper), respectively. There was an increase in the progression to blastocyst for 1-5 embryos per droplet, compared to 10 and 20 (6.6-24.2% vs. 39.2-43.3%, P0.05). For the tested free embryos, those cultured with helpers had increased blastocyst development over their control counterparts (39.3-49.5% vs. 6.6-24.2%, P0.05). When the number of embryos per droplet was 10 or 20, blastocyst percentage was similar (39.2-49.5%, P0.05). In Experiment 2, addition of an agarose chip to the culture medium did not significantly affect development to the blastocyst stage. In Experiment 3, after fertilizing OPU oocytes with sorted X-sperm, a group of three cleaved embryos were cultured in a droplet with either seven helpers (3 + 7) or alone (3 + 0). Blastocyst development of OPU oocytes in the 3 + 7 group was 37.1%, higher than that in the 3 + 0 group (11.8%, P0.05). In conclusion, limited numbers of OPU/IVF oocytes had competent development when cultured with helpers (embedded in agarose to provide physical separation).

The primary objective was to elucidate ovarian follicular dynamics and hormonal profiles in nulliparous heifer (HE; n = 11 ) and mixed-parity (MP; n=10 ) Mediterranean Italian water buffaloes (Bubalus bubalis) following an estrus synchronization protocol. Both groups received a progesterone releasing intravaginal device (PRID) implant for 10 days; a luteolytic dose of synthetic prostaglandin was given 7 days after PRID insertion. Daily ultrasound monitoring and collection of blood to determine plasma concentrations estradiol and progesterone started 1 day after PRID removal and lasted for 55 and 65 days in HE and MP buffaloes, respectively. Data analysis was restricted to the first 5 days after PRID removal and to one estrus cycle following induced ovulation. The HE buffaloes were not inseminated and only one ovulated within 5 days after PRID removal; the remainder ovulated between 8 and 48 days after PRID removal (except one in which ovulation was never detected). All HP buffaloes were inseminated 72, 96 and 120 h after PRID removal; seven buffaloes ovulated within 5 days after PRID removal and two were pregnant. Mean diameter of the largest follicle was significantly smaller in HE than MP buffaloes the first 4 days after PRID removal. There was a parity by time interaction ( P=0.0047 ) for plasma progesterone concentrations; progesterone was higher in HE than MP buffaloes 1 day after PRID removal, but the converse was true 2 days after PRID removal. After induced ovulation, HE buffaloes exhibited a one-wave ( n=5; length of cycle, 8-12 days), two-wave ( n=4; range: 20-26 days) or three-wave cycle ( n=1; 25 days). In contrast, all non-pregnant MP buffaloes ( n=8 ) had a two-wave cycle (range: 19-25 days). For buffaloes with two-wave cycles, the growth rate and diameter of the largest follicle was significantly smaller in HE than MP buffaloes for both the first follicular wave (1.3mm versus 1.7 mm per day and 10.5 mm versus 13.3 mm, respectively) and the second follicular wave (1.0 mm versus 1.3 mm per day and 11.0 mm versus 13.8 mm). In conclusion, there were many significant morphological and endocrine differences between HE and MP buffaloes.

At the time of AI following Ovsynch protocol, a total of 51 buffaloes were randomly divided in a first group (n = 30) subjected to conventional AI into the uterine body with 20 million non-sex sorted frozen-thawed spermatozoa, while a second group (n = 21) was inseminated near the utero-tubal junction (UTJ) ipsilateral to the ovary carrying the preovulatory follicle with 2.5 million live (4 million total) sex-sorted frozen-thawed spermatozoa. The semen used for flowcytometric sorting was collected and processed on a farm in Italy, and then shipped to a laboratory in Germany. Eleven buffaloes were inseminated with X-chromosome bearing spermatozoa and 10 with Y-chromosome bearing spermatozoa. Conception rates after conventional and UTJ inseminations were 43.3% (n = 13) and 42.8% (n = 9) respectively (p = 0.97). Eight of the nine foetuses obtained after insemination with sexed spermatozoa corresponded to the sex as predicted by the cell sorting procedure (five male and four female foetuses by ultrasound vs six male and three female foetuses by cell sorting). In conclusion, for the first time buffalo semen has been successfully subjected to procedures for flowcytometric sperm sorting and freezing. Low doses of sexed spermatozoa have been deposited near the UTJ giving conception rates similar to those of conventional AI with full dose.

The Japanese Black cattle (Wagyu) plays a significant part in the Japanese beef industry because it is numerically the largest breed group and comprises ~93% of the national purebred beef cow herd. Japanese Black cattle are genetically predisposed to intense marbling and to a high percentage of unsaturated fat resulting in a meat characterised by both high quality and price on the market. Like other breeds, genetic improvement and production traits in Wagyu can be fostered by the implementation of traditional reproductive strategies such as multiple-ovulation embryo transfer. A multiple-ovulation embryo transfer case study was performed in a leading production centre in Hainan island, South China. Donors (n = 40) were split into 2 groups receiving either a total dose of 400 mg of FSH Folltropin (FSH USA; Folltropin Bioniche Inc., USA; n = 24), or 10 mg of an FSH formulation produced from the Institute of Zoology of the Chinese Academy of Sciences (FSH CHN; n = 16). In both cases, dosages were equally distributed over a 4-day administration schedule. Both donors and recipients (198 heifers) were synchronized for fixed-time embryo transfer (ET) and AI, respectively, by adopting the Ovsynch protocol. Such protocol consists of GnRH administration at Day 0, followed by prostaglandin administration at Day 7 and a second administration of GnRH at Day 9. Artificial insemination was performed on donor animals at 12 and 24 h from the last GnRH administration, whereas for recipient synchronization receiving fresh embryos, the second GnRH administration was given at the time of second AI on donors, and ET was performed 7 days following the first AI. Final synchronization at the time of ET, judged by the ultrasonic presence of a functional corpus luteum, was 53% (105/198). The following parameters for FSH USA and FSH CHN were found to be not significantly different (Student's t; mean ± s.e.): i) ovulations (10.5 ± 1.2 v. 8.5 ± 1.2; P = 0.2); ii) embryos (6.3 ± 1.2 v. 5.1 ± 1.1; P = 0.5), and iii) embryos from ovulated donors (6.8 ± 1.3 v. 5.8 ± 1.1; P = 0.6). Recovered embryos from the 2 groups were also not different: i) degenerated embryos (1.0 ± 0.4 v. 0.8 ± 0.2; P = 0.6); ii) morula (4.0 ± 0.8 v. 2.6 ± 0.5; P = 0.1), and iii) early blastocysts (4.0 ± 1.2 v. 3.3 ± 0.5; P = 0.6). Blastocysts were recovered only from donors treated with FSH USA. Out of 233 recovered embryos, 34 were transferred as fresh and 71 as frozen/thawed. Pregnancy rate at 60 days following ET for fresh and frozen/thawed embryos was 47.1 and 35.2%, respectively (P = 0.2). Within frozen embryos, pregnancy rates derived from transferred morulas and blastocysts were 25 and 45.5% (P = 0.07). When considering the two sources of hormones, overall pregnancy rates were similar between the two groups (28/71, 39.4% v. 13/34, 38.2%; P = 0.9). Finally, pregnancy rates from the transfer of fresh embryos (9/21, 42.8% v. 7/13, 53.8%; P = 0.6) and frozen/thawed embryos (19/50, 38% v. 6/21, 23.8%; P = 0.6) were also not different. In conclusion, all parameters in this study did not differ between the 2 sources of FSH; however, a lower incidence of degenerated embryos and a higher pregnancy rate following transfer of frozen/thawed embryos occurred when FSH USA was used in donor animals.

An ultrasound assessment of follicle turnover following two different protocols for synchronization of oestrus and ovulation, as well as an assessment of achieved synchronization between ovulation and AI and conception rates in nulliparous and pluriparous buffaloes were carried out during months of increasing day length. Nulliparous buffaloes (n = 30) were subjected only to Ovsynch protocol whereas pluriparous buffaloes (n = 31) were assigned to Ovsynch (n = 14) or to PRID-pregnant mare serum gonadotrophin (PMSG) (n = 17) protocol according to the presence of functional CL confirming cyclic and acyclic conditions. Ultrasound examination of ovarian follicular dynamics at critical days in the course of synchronization treatments was employed to monitor the fate of the largest available follicles at the beginning of treatments. Such available dominant follicle would persist throughout the protocol as ovulating follicle (no-follicle shift) or would regress giving way to a new follicle to become dominant and ovulate (follicle shift). Furthermore, ultrasound monitoring would determine the degree of synchronization of ovulation and final outcome represented by pregnancy rates. Pregnancy rate following Ovsynch protocol was 40% (12/30) and 42.8% (6/14) in nulliparous and pluriparous buffaloes respectively (p = 0.8575). Most ovulations were synchronized and recorded at AI and the following day in nulliparous (24/30; 80%) and pluriparous (12/14; 85.7%) buffaloes respectively (p = 1.000). A follicle shift was recorded in 14 of 30 (46.6%) and 11 of 14 (78.5%) in nulliparous and pluriparous buffaloes respectively (p = 0.0466). Among established pregnancies: eight derived from follicle shift (66.6%) and four from no-follicle shift (33.3%) in nulliparous buffaloes, p = 0.0729 whereas in pluriparous buffaloes five (83.3%) derived from follicle shift and one from no-follicle shift (16.6%), p = 0.6154. Collectively, from 18 pregnancies in nulliparous and pluriparous buffaloes following Ovsynch protocol, 13 derived from follicle shift (72.2%) and five from no-follicle shift (27.7%), p = 0.0860. Pregnancy rate in pluriparous buffaloes following PRID-PMSG protocol was 70.5% (12/17). The majority of ovulations were synchronized and recorded at first, second AI and following day (13/17; 76.4%). A follicle shift occurred in 15/17 buffaloes (88.2%) and among the 12 recorded pregnancies, 11 derived from follicle shift (91.6%), p = 0.5147. In conclusion, pregnancy rates following Ovsynch protocol were similar in nulliparous and pluriparous cyclic buffaloes. A progestagen treatment on acyclic buffaloes but still displaying some ovarian follicular dynamics, resulted in significantly higher pregnancy rate compared with Ovsynch (p = 0.0376). According to the time of scheduled AI, a high degree of synchronized ovulations were recorded following the implementation of both protocols.

The objective of this study was to monitor ovarian function in postpartum primiparous and pluriparous Mediterranean Italian buffaloes (Bubalus bubalis) during months of increasing daylength. Ovarian ultrasound monitoring was carried out for a total of 60 days from calving in 10 primiparous and 10 pluriparous buffaloes. Progesterone was determined from calving until a week after first postpartum ovulation. The study was undertaken during months of increasing day length. Time required for complete postpartum uterine involution was 31 +/- 1.0 and 33 +/- 1.3 days in primiparous and pluriparous buffaloes respectively (P = 0.1). The first postpartum ovulation was recorded on 4 primiparous and 8 pluriparous buffaloes (P = 0.16). Time for first postpartum ovulation to occur was 25.5 +/- 6.9 and 15.5 +/- 1.3 days in primiparous and pluriparous buffaloes, respectively (P = 0.07). Overall, 8 of the 12 first postpartum ovulations (66.6%) occurred in the ovary contra-lateral to the one bearing the gravidic CL, one out of 4 in primiparous and 3 out of 8 in pluriparous buffaloes (P = 1.0). Following a first postpartum ovulation, 3 primiparous and 4 pluriparous buffaloes displayed a complete wave of follicular development leading to a new ovulation. Ovulation following parturition was not recorded in 6 primiparous and two pluriparous buffaloes for the 60 days of ultrasound monitoring. Growth rate (mm/d) and largest size (mm) of first postpartum ovulating follicle was 0.95 +/- 0.18 and 1.07 +/- 0.07 (P = 0.4), and 13.5 +/- 0.8 and 14.1 +/- 0.4 (P = 0.4) in primiparous and pluriparous buffaloes, respectively. Following calving, the total number of available antral follicles (or =2 mm) declined gradually towards the end of the study period. Follicles greater or equal to 3 mm in diameter on the contrary showed a prominent increase in the first 2 weeks from calving. The number of follicles greater or equal to 3 mm in diameter was significantly higher in the ovary contra-lateral to the one bearing the gravidic CL. A balance in the number of such follicles was reached toward the end of the first month. In conclusion, although some follicular activity was recorded in the ovaries of all buffaloes, true postpartum resumption of cyclicity in the months of increasing daylight hours was delayed in the majority of animals.

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 and200kg) 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 (P0.05). With the exception of one calf, some minor follicular waves (short waves or SWs; 1.6+/-1), lasting10 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, P0.05). 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, P0.05). 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.

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.

Recently, the inclusion of sexed semen technology under field conditions in Mediterranean buffalo heifers has already proved feasible, resulting in pregnancy rates similar to what is ordinarily achieved with conventional nonsexed semen. A seasonal species within the latitudes currently reared in Italy, buffaloes tend to perform better reproductively during the period of the year of decreasing day length. In this study, sexed and nonsexed semen from four bulls has been used in two farms in the south of Italy. Following Ovsynch synchronization, 152 pluriparous buffaloes selected after screening for good general and reproductive health were subjected to AI in two periods of the year: (1) September to October and (2) January to February. Sexed and nonsexed semen was randomly used via AI with a total concentration of 2 × 106 and 20 × 106 spermatozoa per single straw, respectively. Pearson's chi-square test and Fisher's exact test were used for evaluation of data. Neither a bull nor farm effect on pregnancy rates was detected and therefore data for these two variables were pooled. Overall, combining the data from the two periods investigated, the use of sexed semen resulted in pregnancy rates at 28 days following AI similar to the use of nonsexed semen: 50/79; 63.2% vs 44/73; 60.2% (P = 0.70), respectively. Confirmation of pregnancy at 45 days post-AI also resulted in similar rates for both sexed and nonsexed semen: 49.3% vs 45.2% (P = 0.60), respectively. When individually considering each of the two periods investigated, the use of sexed semen in the months of September to October gave pregnancy rates at 28 days post-AI similar to nonsexed semen: 54.5% vs 56% (P = 0.88) and similarly at 45 days post-AI: 45.4% vs 48.7% (P = 0.75). A similar trend was observed in the months of January to February at 28 days post-AI for sexed and nonsexed semen: 74.2% vs 65.6% (P = 0.43) and similarly at 45 days post-AI: 54.2% vs 40.6% (P = 0.26). The use of sexed semen did not influence the pattern of late embryonic mortality (LEM) checked at 45 days post AI, whereas the season was a significant variable affecting LEM. In fact, when combining data for both sexed and nonsexed semen, LEM rate was found to be higher (P = 0.05) during the months of January to February (15/47; 31.9%), with the following individual values for sexed semen 7/26 (26.9%) and nonsexed semen 8/21 (38.1%), when compared with LEM rate during the months of September to October (7/47; 14.8%), with the following individual values for sexed semen 4/24 (16.6%) and nonsexed semen 3/23 (13.0%). In conclusion, although pregnancy rates were reported to be higher in the months of January to February when compared with the months of September to October, the former months seemed to affect more profoundly LEM for both sexed and nonsexed semen.

The main scope of this study was to evaluate the likelihood of a helper effect of agar-embedded cleaved embryos on a low number of free embryos at a similar stage of development within the same culture droplet. Such an improved system could be beneficial within ovum pickup/in vitro embryo production (OPU/IVEP) combined protocols whenever a low number of OPU-derived cleaved embryos are produced per donor. Oocytes were recovered from abattoir ovaries, and after in vitro maturation (IVM) and in vitro fertilization (IVF), presumptive zygotes were deprived of cumulus investment and allocated into culture droplets for 24 h. At 48 h from IVF, 4- to 8-cell cleaved embryos were randomly allocated into a control and a treatment group. Control groups consisted of 1, 3, 5, and 10 embryos, respectively, in 50-μL droplets. Treatment groups consisted of 1, 3, and 5 free embryos with the addition of 9, 7, and 5 embryos, respectively, at a similar stage of development embedded in agar chips, so as to reach a total number of 10 cleaved embryos in each culture droplet. Culture was performed for both the control and treatment groups in SOF medium droplets covered with mineral oil, with the supplementation of essential and nonessential amino acids in a controlled gas atmosphere consisting of 5% CO2, 7% O2, and 88% N2 at 39°C. Final embryo output was checked at Day 7 from IVF. When considering only free embryos, the difference in progression to blastocyst development was highly significant between the control and treatment groups: 1) group 1 v. 1 + 9: 6.6 v. 84.3% (P = 0.00000); 2) group 3 v. 3 + 7: 11.1 v. 41.3% (P = 0.00001); 3) group 5 v. 5 + 5: 24.4 v. 42.2% (P = 0.00001). Rate of blastocyst development in the control group containing 10 cleaved embryos was not significantly different from free cleaved embryos in the 3 + 7 (39.2 v. 41.3%, P = 0.71) and 5 + 5 treatment groups (39.2 v. 42.2%, P = 0.54), but was significantly lower when compared with the 1 + 9 treatment group (39.2 v. 84.3%, P = 0.000). For 1, 3, 5, and 10 control group embryos, the numbers of replicates and total cleaved embryos used (n) were 30 (n = 30), 27, (n = 81), 27 (n = 135), and 39 (n = 390), respectively. For the 1 + 9, 3 + 7, and 5 + 5 treatment group embryos, the numbers of replicates and total cleaved embryos used were 32 (n = 32), 29 (n = 87), and 27 (n = 135), respectively. In conclusion, a beneficial effect of agar-embedded embryos on the development of free embryos within the same culture droplet was shown. A striking improvement in late-stage embryo development was particularly evident when considering the 1 v. 1 + 9 control and treatment groups. These results may foster a different strategic approach in in vitro culture to enhance embryo development from highly valuable donors.

The main objective of this study was to compare pregnancy and calving rates following transfer of in-vitro-produced fresh river and F1 (river × swamp) buffalo embryos in recipients synchronised by Ovsynch protocol or following natural oestrus. River embryos were produced from cumulus–oocyte complexes (COCs) derived by ovum pick up (OPU) on 40 Murrah and Nili-Ravi donor buffaloes over a twice-weekly collection schedule for 120 single OPUs. F1 embryos were produced by fertilisation of swamp COCs recovered from abattoir ovaries coincubated with river sperm cells. Both groups of embryos were produced following the same protocol for in vitro production. With regard to the OPU source of COCs, 923 antral follicles were punctured and 647 COCs were recovered (70%). From 462 selected COCs for IVM, 257 (55.6%) cleaved zygotes were recorded leading to 93 blastocysts (20.1%). In total, 590 swamp COCs were aspirated from abattoir ovaries and 476 were selected for IVM leading to 270 (56.7%) cleaved zygotes and resulting in 137 blastocysts (28.8%). River and F1 embryos were transferred between Day 6 to 7 of in vitro development, corresponding to blastocyst–expanding blastocyst, into F1 recipients synchronised by Ovsynch and swamp buffaloes following natural oestrus, respectively, each of them receiving two embryos. According to palpation per rectum of the ovaries at the time of embryo transfer, 26 of the 47 (55.3%) F1 recipients synchronised by Ovsynch were considered suitable for transfer, resulting in seven pregnancies (26.9%) and four calvings (15.3%) owing to three abortions occurring between 2 and 3 months of pregnancy. In total, 29 swamp recipients following natural oestrus were judged suitable as recipients, resulting in 12 pregnancies (41.4%) and 10 calvings (34.5%) owing to two abortions at 2 and 3 months of gestation respectively. Pregnancy and calving rates following transfer of river and F1 embryos were similar. Likewise, weight at birth of calves derived from transfer of river and F1 embryos was not different: 30.5 ± 1.4 and 32.9 ± 2.4 respectively. Pregnancy and calving rates following AI in a group of river and swamp buffaloes considered for reference in this study were similar to recipients carrying in-vitro-produced embryos. Collectively, no apparent postnatal complications were recorded in resulting live calves.

Following semen collection, the nonspecific bacterial count is usually kept low by addition of antibiotics to the extender. Ordinary protocols for semen processing in preparation for IVF are characterized by a washing procedure, followed by spermatozoa selection by either swim-up or Percoll gradient. In this study, commercially available bovine semen to be used for IVF was found to be heavily contaminated with Escherichia coli and Leclercia adecarboxilata despite the addition of tylosin (5 mg/100 mL), gentamicin (25 mg/100 mL), spectomycin (30 mg/100 mL), and lincomycin (15 mg/100 mL) to the extender. Bacterial concentrations varied among straws examined (n = 7), ranging from 10 � 4 UFC mL-1 to 10 � 8 UFC mL-1. Noncontaminated semen from the same valuable bull, although derived from a different semen collection, was known to be used with great efficiency for IVF in a different laboratory at a very low concentration (i.e. 350 000 sperm mL-1). Therefore, to rescue the fertilizing capacity of such contaminated semen, an improved treatment was applied and compared with the ordinary semen treatment. From the previous use of semen from the same bull, a concentration of 2 million sperm/mL-1 and 20 �g mL-1 of heparin was chosen for IVF. During ordinary treatment, the semen was washed using 6 mL of fertilization medium into a 15-mL tube and subjected to centrifugation at 800 rpm for 5 min. After removal of the supernatant, the resulting pellet was layered under 1 mL of the same medium and left for 45 min in an incubator for swim-up. The supernatant was moved into a new 15-mL tube for additional centrifugation. The newly formed pellet was then resuspended for the final count and co-incubation with oocytes. With such a procedure, the bacterial contamination was found for 3 consecutive replicates in culture droplets starting at Days 3 to 4 post-IVF. In the course of 5 additional replications, the semen treatment was improved by doubling the volume for initial washing to 12 mL of fertilization medium. The resuspended pellet following the first centrifugation was equally partitioned into 6 round-bottomed tubes and left for 60 to 80 min in an incubator for swim-up. The supernatant was then moved into a new 15-mL tube for additional centrifugation and a final count. The IVF medium was supplemented with 100 IU mL-1 of penicillin G in both treatments. Following the ordinary and improved semen treatment, a large number of sperm cells were found agglutinated head-to-head. A subsequent embryo culture was performed for both treatments in SOF containing 0.025 mg mL-1 of kanamycin sulfate. When using sperm and heparin concentrations at 2 million and 20 �g mL-1, respectively, under the improved semen treatment, cleavage was 84.8%. The rates of blastocyst production at Day 7 from used cumulus-oocyte complexes and from cleavage were 29.5 and 35.9%, respectively. In conclusion, the improved washing procedure rescued valuable but heavily contaminated semen for use in IVF procedures.

The use of AI in the buffalo species is still marginal due to traditional lower conception rates when compared to cattle. However recently a number of studies in this field have revealed a promising increase in the efficiency of synchronization protocols for AI linked to more acceptable pregnancy rates. The possibility of using lower spermatozoa concentration of high quality buffalo bulls for AI without reduction in pregnancy outcome can be an additional offset, especially if such spermatozoa can be sexed and used for better reproductive management in buffalo farms. Within this conceptual framework, a new artificial insemination device for semen deposition near the utero-tubal junction (UTJ) in cattle (Ghent device), developed at the University of Ghent (Belgium), has been used in this study. The Ghent device is made of disposable materials and consists of 2 hollow plastic tubes, wherein a catheter filled with semen is introduced. The outer tube is completely rigid, while the inner tube consists of a rigid caudal end and a flexible cranial tip. The outer plastic tube can move independently from the inner tube. Once the insemination device is introduced into the uterine body, the inner tube with its flexible tip is moved forward. The flexible tip makes it possible to follow the curvature of the contractile uterus of the estrous cow, but excludes the use of Cassou straws. The semen is then expelled from the catheter by means of 0.1mL of air followed by 0.6mL of physiological saline solution. To assess the efficacy of the new Ghent device, 67 buffalo cows (Bubalus bubalis) were inseminated during a field trial. Two different insemination methods were used: (1) insemination with the conventional insemination device in the uterine body, and (2) insemination with the Ghent device near the utero-tubal junction ipsi-lateral to the site of ovulation. Artificial insemination was performed twice at 72 and 96hrs after administration of prostaglandins to buffaloes bearing a functional corpus luteum as recorded during ultrasound monitoring. Conventional inseminations were performed with full (16–20×106) and half (8–10×106) insemination doses of frozen-thawed semen, while UTJ-inseminations were performed with full, half and quarter (4–5×106) insemination doses. When inseminations were performed with the conventional insemination device, halving the insemination dose resulted in a non-significant decrease of conception rates from 53% (8/15) to 42% (8/19). However, no difference in conception rates was observed when UTJ-inseminations were performed with a full, half or quarter standard insemination dose: 50% (6/12), 45% (5/11) and 50% (5/10), respectively. Despite the limited number of inseminations performed, this preliminary field trial demonstrates that the Ghent device is suitable for the insemination of buffaloes under field conditions, and that UTJ inseminations can be performed with only one-fourth of the standard insemination dose without a reduction in conception rates.

In cattle and buffaloes resumption of ovarian activity, as expressed by first post partum ovulation and follicle turnover leading to periodic estrus cycles and ovulations, can be affected by many factors among which uterine infections and dietary supplementation play a leading role. In addition, buffaloes are considered seasonal animals characterized by a tendency to reproduce in correspondence to a decrease of day length. This aspect may further affect their reproductive efficiency by delaying or interrupting ovarian activity if calving occurs in transitional (i.e., from end winter into beginning of spring) or unfavourable (spring-summer) periods of the year, when an increase of daylight hours occurs. The aim of this study, carried out in the months of February to May, was to understand the effect of increasing daylight hours over resumption of postpartum ovarian follicular dynamics and estrus cycles. Mixed parity Mediterranean Italian buffaloes (n=20) were monitored daily by ultrasonography from calving until first postpartum ovulation and following cycle or until 60 days if no ovulations had been recorded. Ultrasound monitoring was performed by using a 7.5MHz linear-array probe and an Aloka SSD-500 monitor. No pharmacological intervention was allowed during the course of the study and animals were kept under similar feeding and farm management conditions. All animals exhibited a mild to moderate uterine inflammation (n=13; 65%) or aspecific infection (n=7; 35%) after calving. Infections were manifested as purulent discharge and as ultrasound detection of highly echogenic debries in the uterine lumen. Only one animal retained pathological accumulation in both horns for the entire duration of the study. Among the 20 buffaloes under study, 12 (60%) exhibited postpartum ovulations, ranging from 9 to 45 days. In eight buffaloes (40%), such ovulations occurred in the ovary contralateral to the ovary bearing the gravidic CL. Following postpartum ovulations, seven buffaloes (35%) underwent a complete cycle leading to ovulation. Three of such cycles (15%) culminated with a second ovulation in the same ovary where post partum ovulations had occurred. Among the full cycles following postpartum ovulations, four of them were one-wave, with a range of 8 to 19 days, whereas the remaining three cycles were two-wave with a range of 17 to 22 days. Eight buffaloes (40%) were reported in anovulatory condition following parturition, although in four of them both ovaries displayed a typical follicle turnover. In conclusion, resumption of ovarian activity and reproductive efficiency in water buffaloes can be affected by increasing day length as evidenced by a high rate of postpartum anovulatory conditions and reduced follicle turnover.