Reproduction, Fertility and Development

Temporal dynamics of ram sperm binding and survival during 48-h coculture with oviducal epithelial cells

2012-01-31T19:27:08Z

Temporal dynamics of ram sperm binding and survival during 48-h coculture with oviducal epithelial cells Following insemination, ram spermatozoa bind to oviducal epithelial cells (OEC) in vivo and remain viable for several hours before fertilisation. In the present study, we investigated whether OEC monolayers reproduce this effect in vitro, performing an analysis of ram sperm binding and survival over an extended (48 h) period at 39�C. We wanted to determine whether the reproductive cycle phase and/or oviducal region would influence ram sperm binding and survival in coculture with OEC and whether reproductive and non-reproductive epithelial cells bound and maintained the viability of ram spermatozoa equivalently. Oviducts were separated into groups based on their ovarian state (follicular or luteal) and then divided into two parts (isthmus and ampulla) for OEC isolation. Sheep kidney epithelial cells (Madin-Darby ovine kidney; MDOK) were purchased commercially. Reproductive cycle phase, but not oviducal region, affected sperm binding to OEC. Although more spermatozoa bound to luteal OEC than to follicular OEC at 1 h, at 24 h follicular OEC had bound more spermatozoa than luteal OEC. Generally, spermatozoa that were bound to OEC and MDOK had enhanced viability at each of the time points investigated (1, 6, 24 and 48 h), but the viability of the OEC-bound spermatozoa was greater than that of the MDOK-bound spermatozoa at 48 h. In conclusion, ram sperm-epithelial cell interactions are temporal, dynamic and depend on the origin of the epithelial cells.

Alteration in testicular cell components following transiently induced ischaemia in prepubertal bulls

2012-01-31T19:26:38Z

Alteration in testicular cell components following transiently induced ischaemia in prepubertal bulls The aim of the present study was to evaluate transient testicular ischaemia (induced using elastrator bands) in Jersey calves on testicular morphology and development. Treatments (at 27 � 5 days of age) consisted of control (0 h banding) and banding for 2, 4 or 8 h (n = 4 in each group). After castration (at 60 � 5 days of age), the right testis was used for calculation of cell components per testis according to the point-counting method. Bodyweight (59.8 � 6.2 kg) and scrotal circumference (SC) at banding (9.1 � 0.2 cm) did not differ between groups. Fresh testis weight, scrotal temperature immediately before band removal and daily SC growth were decreased in ischaemic (4 and 8 h) testes compared with controls (P < 0.05). In addition, the number of Sertoli and Leydig cells was significantly reduced in the 8 h ischaemic treatment group (P < 0.05). Transiently induced ischaemia significantly decreased the number of germ cells in the 8 h ischaemic treatment group (13 � 5 X 106 cells) compared with the 0, 2 and 4 h ischaemic treatment groups (38 � 6, 32 � 6 and 33 � 5 X 106 cells, respectively; P < 0.05). These results suggest that transiently induced ischaemia for 8 h significantly decreases the number of germ, Sertoli and Leydig cells in prepubertal testis.

Technical Note: Effect of density gradient centrifugation with trypsin on the in vivo fertilising capability of bovine spermatozoa

2012-01-31T19:26:08Z

Technical Note: Effect of density gradient centrifugation with trypsin on the in vivo fertilising capability of bovine spermatozoa The present study investigated the effect of a novel density gradient centrifugation (DGC) treatment using recombinant trypsin on the in vivo fertilising capability of bovine spermatozoa compared with a standard method. In Trial 1, semen collected from Boran and Ankole (Bos indicus) bulls was treated either with a silane-coated silica particle colloid formulated for humans with a recombinant trypsin or processed using a standard method (dilution in an egg yolk-based diluent). Semen processed by the two methods was used to artificially inseminate (AI) superovulated cattle. Day 7 embryos were flushed and assessed for fertilisation rates and embryo quality. Trial 2 used a trypsinised silane-coated silica particle colloid formulated specifically for bovine semen. Trial 1 resulted in significantly higher fertilisation rates using the trypsinised human DGC treatment than cows inseminated using the standard method (75.2% v. 67%, respectively; P < 0.01), but the numbers of transferable-quality Day 7 embryos did not differ between the two groups (P > 0.05). Results for Trial 2 indicated that cows inseminated with the trypsinised bovine DGC treatment had significantly increased fertilisation rates compared with the standard method (88.4% v. 63.1%, respectively; P < 0.01) and had significantly higher numbers of transferable-quality embryos (70.3% v. 51.8%, respectively; P < 0.01). In summary, bovine sperm treatment before AI by DGC and recombinant trypsin increases fertilisation rates and can result in more transferable-quality embryos compared with standard methods.

A unique method to produce transgenic embryos in ovine, porcine, feline, bovine and equine species

2012-01-31T19:25:37Z

A unique method to produce transgenic embryos in ovine, porcine, feline, bovine and equine species Transgenesis is an essential tool in many biotechnological applications. Intracytoplasmic sperm injection (ICSI)-mediated gene transfer is a powerful technique to obtain transgenic pups; however, most domestic animal embryos do not develop properly after ICSI. An additional step in the protocol, namely assistance by haploid chemical activation, permits the use of ICSI-mediated gene transfer to generate transgenic preimplantation embryos in a wide range of domestic species, including ovine, porcine, feline, equine and bovine. In the present study, spermatozoa from five species were coincubated with pCX-EGFP plasmid and injected into metaphase II oocytes. The chemical activation protocol consisted of ionomycin plus 6-dimethylaminopurine. We detected high proportions of fluorescent EGFP embryos for all five species (23-60%), but with a high frequency of mosaic expression (range 60-85%). To our knowledge, this is the first study to produce exogenous DNA expression in feline and equine embryos. Chemical activation reduces the lag phase of egfp expression in ovine embryos. Our results show that this unique method could be used to obtain ovine, porcine, feline, bovine and equine transgenic preimplantation embryos.