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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.

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.

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.