Studies on cryopreservation of semen of farmed red deer (Cervus elaphus) and fallow deer (Dama dama)

Abstract

Studies presented in this thesis investigated alterative extender-cryoprotectant combinations for semen preservation in red deer (Cervus elaphus) and fallow deer (Dama dama). Semen was collected by electroejaculation under anaesthesia and pooled from four red deer stags and four fallow deer bucks. The ejaculated semen was diluted to a final concentration of 100x10⁶ sperm/ml in sodium citrate, tris citrate, skim milk, lactose and ram synthetic based extenders, with glycerol, propan-1, 2-diol (PROH) and dimethyl sulphoxide (DMSO) cryoprotectants at two concentrations, undiluted (U) and half dilution (0.5D). Semen was frozen in 0.25 ml straws in an automated chamber freezer and stored immersed in liquid nitrogen. In vitro laboratory evaluation of frozen-thawed semen involved the recording of motility and progressive motility of sperm by phase contrast microscopy over a 4 hour incubation period at the equivalent rectal body temperature of female red and fallow deer. Post-thaw (t=0) acrosomal status and sperm morphology were determined by histology using phase contrast optics.

Red deer semen gave the highest post-thaw motility (56.7±8,4%) and incubation performance with tris citrate-glycerol (0.5D) extender. Glycerol was the overall optimum cryoprotectant at the undiluted concentration for both deer species, the above being the only exception. Lactose glycerol (U) was the superior extender buffer (p<0.001) for the maintenance of acrosomal integrity of both red and fallow deer semen. Skim milk: glycerol (U) gave the highest motility (76.7±8.0%) (P<0.05) for the post-thaw and initial 2 hours of the incubation period for fallow deer. There was no treatment effect on sperm morphology for both deer species.

The second part of the thesis involved in vivo evaluation of the two optimal extender-cryoprotectant combinations, with the sodium citrate-glycerol (U) extender as the basis for comparison. A total of 205 mixed aged red deer hinds across 3 farms were randomly allocated into 3 treatment groups. All hinds were synchronised with progesterone-containing controlled internal drug release (CIDR) devices for 12 days, with device renewal on Day 8. A total dosage of 200 iu pregnant mare serum gonadotrophin (PMSG) was administered at device withdrawal and laparoscopic intrauterine AI was performed 56 h post-CIDR device withdrawal. Conception rates, assessed by rectal ultrasonography on Day 45, were similar across the three extender treatments, sodium citrate (50.3±6.5%), tris citrate (50.3±6.5%) and lactose (40.5±6.5%). The poor performance of the tris citrate extender on one farm (33.3%) and lactose extender on another (37.2%) resulted in a farm by treatment interaction (P<0.10).

For fallow deer, a total of 278 mixed aged does across 3 farms was randomly allocated into 3 treatment groups, sodium citrate, skim milk: and lactose. Oestrus was synchronised using CIDR devices for 14 days, with laparoscopic intrauterine AI performed 70 h post-CIDR device withdrawal. Conception rates ranged from 64.9±4.9% (sodium citrate) to 71.2±4.6% (lactose) (P>0.10), with a genotype effect (P<0.05) favouring the ¼ Mesopotamian genotype (75.7±6.5%) versus the European does (61.1±4.9%). There was an inseminator effect (P<0.05) with one inseminator achieving very high conception rates with the ¼ Mesopotamian does (83.3±5.8%).

In summary, the evaluation of semen in vitro identified alternate extender clyoprotectant combinations which improved semen preservation for red and fallow deer, however these differences were not reflected in conception rates to AI.