G.W. Asher

Storage of semen and artificial insemination in deer

Methods of collection and freezing of semen of some deer species and aspects of controlled reproduction associated with the use of frozen-thawed semen by artificial insemination (AI) are discussed.

EMBRYO TRANSFER IN DEER

Abstract The rapid growth in deer farming and interests in the conservation of various deer species have stimulated development of embryo transfer procedures. The protocols developed are based on those used in sheep and cattle and generally involve surgical embryo recovery following FSH-induced superovulation. Data are limited, but pregnancy rates of around 3.0 per donor female for red deer, and around 1.5 for fallow deer are being achieved.

New developments reproductive technologies in deer

In vitro embryo production is the platform for advanced reproductive technologies, such as cloning. The in vitro embryo production system developed for farmed red deer (Cervus elaphus) evolved along similar lines to that pioneered by other domestic species researchers. However, applying existing in vitro embryo production methods from these other species resulted in limited success and has necessitated developing a species-specific methodology for red deer based on the their physiology. Analysis of oviduct fluid led to the development of a semi-defined fertilization and culture media system, Deer Synthetic Oviduct Fluid (DSOF), which resulted in successful culture of red deer embryos to the blastocyst stage. Transvaginal ultrasound-guided ovarian examination and ovum pickup has enabled the study of seasonality constraint and propagation from selected female genetics, respectively. During the 4-month breeding season (April-July), 15% of cleaved oocytes developed to blastocysts, whereas no blastocysts developed from oocytes collected after July. The process of developing an in vitro embryo production system for farmed red deer may serve as a beneficial model for the propagation of endangered cervine species.

Environmental constraints on reproductive performance of farmed deer

Abstract Optimum reproductive efficiency of farmed deer is often constrained by apparent maladaptation of wild cervids to a given farm environment or components of that environment. Seasonal breeding amongst forest-fringe species of northern temperate origin (e.g. red deer, fallow deer) benefits the species within its natural range, but often creates misalignment in southern temperate zones between the high nutritional demands of lactation in summer and the earlier peak of pasture production and quality occurring in spring. This can lead to increased calf mortality, decreased calf growth rates and depressed dam liveweights. Much emphasis has been placed on manipulating either pasture production patterns or the timing of birth to create better synchrony between the two. The latter largely involves artificial manipulation of the timing of conceptions (e.g. melatonin treatment), but there may also be options for genetic selection of deer for earlier calving patterns. Tropical cervid species farmed in temperate environments can exhibit either aseasonal reproduction (e.g. chital deer) or autumn-wintering calving patterns (e.g. rusa deer), leading to high mortality of neonates (hypothermia, predation) under uncontrolled breeding management. However, there are simple management options to synchronise and shift calving patterns to better align tropical species to temperate environments. The effects of chronic and acute stress on reproductive efficiency of farmed deer are often discussed but seldom demonstrated. Many farmed deer populations exhibit high pregnancy rates (i.e. > 90% females pregnant), indicating robust reproductive physiology and general habituation to the farmed habitat. Low reproductive rates of pubertal red deer hinds in some herds may be partly indicative of the effects of chronic stress, although other factors such as social facilitation and nutrition cannot be discounted. While species of cervids that are used for farming generally display a high degree of behavioural plasticity, the farm environment and associated management practices may impinge on important components of the social environment. Thus, practices such as controlled breeding (e.g. single-sire mating) and confinement during calving may have effects on reducing overall reproductive potential by disrupting social processes (e.g. dam—calf bonds). This highlights the paucity of knowledge on the complexities of cervid behaviour and their implications in both wild and farmed deer.

Development of in vitro embryo production systems for red deer (Cervus elaphus). Part 1. Effect of epithelial oviductal monolayers and heparin on in vitro sperm motility and penetration of in vitro matured oocytes

In vitro fertilisation (IVF) protocols for red deer have yielded low fertilisation rates, with no embryo development beyond the eight-cell stage when heparin was used as the in vitro capacitation agent. As this low fertilisation rate may result from reduced motility, the present study investigated the use of red deer oviduct epithelial cell monolayers (COEM) and conditioned medium (Cm) from the monolayers to maintain red deer sperm motility in vitro. A second experiment compared the fertilisability of red deer sperm pre-incubated for 4-12h on COEM or for 4h in TALP medium supplemented with 20 microg of heparin.COEM was superior in maintaining red deer sperm motility compared with either Sp-TALP alone or Cm (P<0.05). COEM sustained sperm motility at levels comparable to the initial motility over the 24h period. The motility of sperm incubated in Sp-TALP and Cm was similar and had declined to less than 10% by 4h and no motile sperm were observed by 8h. Overall, the penetration rates of in vitro red deer oocytes were low (5-28%) regardless of sperm treatment. Sperm pre-incubated on COEM penetrated more oocytes than sperm incubated with heparin (P<0.001). Penetration rates were similar for 4-12h pre-incubation of sperm on COEM (P>0.50). Penetration rates were greater across all treatments when both sperm and oocytes were co-incubated for 24h compared to 12h (P<0.001). There were no differences in penetration rates among the four donor stags used in the study. It was concluded that COEM sustains red deer sperm motility in vitro during the 24h observation period. Pre-incubating sperm on COEM does increase sperm penetration rates compared with heparin alone, but at a rate too low and variable to be used on a routine basis. Overall, the penetration rates were comparable to those previously reported for red deer even though differences in heparin concentration, fertilisation systems and stags were used.

Embryo transfer in fallow deer ( Dama dama ): Superovulation, embryo recovery and laparoscopic transfer of fresh and cryopreserved embryos

Multiple ovulation-embryo transfer (MOET) protocols for farmed fallow deer (Dama dama) were investigated in a series of 3 experiments. A total of 37 donors, of either European (D.d. dama ; n = 30) or Mesopotamian hybrid (D.d. mesopotamica x D.d. dama ; n =7) genotype, each received an intravaginal silastic device containing 0.3 g progesterone (CIDR-type G device) for 14 d and injections of 0.5 units ovine FSH (8 x 0.06 unit injections from Days 10 to 14 of device insertion) and 100 IU PMSG (either with the first or last FSH injection). All donors received laparoscopic intrauterine inseminations of fresh semen (50 x 10(6) spermatozoa) from a Mesopotamian sire 36 h after withdrawal of CIDR devices. Embryos were recovered by laparotomy on Day 6 (Day 0 = estrus). Mean ovulation rates for the 3 experiments were 8.1, 9.8 and 7.0, with no effect of PMSG timing (P>0.10). However, embryo recovery rates, albeit low throughout the study (29.6%), were significantly improved with later PMSG administration (33.9 vs 20.1%; P<0.05). Hybrid and European donors performed in a similar manner. A range of embryo development stages was recovered throughout the study. In 2 experiments laparoscopic transfer of embryos to 48 recipient does treated previously with intravaginal CIDR devices for 14 d yielded a total pregnancy rate of 37.5%. In the experiment with fresh embryos, the use of clenbuterol to reduce uterine turgidity resulted in a higher proportion of does conceiving (3/4 ; 75%) compared with that of the untreated does (0/6 , 0%; P<0.05). In the second experiment, in which all the does routinely received clenbuterol, 10/19 (53%) and 5 19 (26%) does conceived following the transfer of fresh and cryopreserved embryos, respectively (P<0.05). While the overall efficiency of the MOET program was low (equivalent of 0.9 to 1.0 surrogate pregnancies per donor), improvements in the recovery rate of transferable embryos have considerable potential for genetic improvement of farm stock and captive propagation of endangered Mesopotamian fallow deer through maternal surrogacy programs.

Genetic influences on reproduction of female red deer (Cervus elaphus): (1) Seasonal luteal cyclicity

This study compared the onset and duration of the breeding season of female red deer (Cervus elaphus scoticus) and its hybrids with either wapiti (Cervus elaphus nelsoni) or Père Davids (PD) deer (Elaphurus davidianus). In Trial 1 (1995), adult red deer (n=9), F1 hybrid wapiti x red deer (n=6) and maternal backcross hybrid PD deer x red deer (i.e., 14 PD; n=9) were maintained together in the presence of a vasectomised red deer stag for 12 months. They were blood-sampled daily or three times weekly so that concentration profiles of plasma progesterone could be used to identify the initiation, duration and cessation of luteal events. There was clear evidence of luteal cyclicity between April and September, with the transition into breeding associated with an apparent silent ovulation and short-lived corpus luteum (i.e., 6-12 days) in every hind. A significant genotype effect occurred in the mean time to first oestrus (P<0.05), with wapiti hybrids and 14 PD hybrids being 9 and 5 days earlier than red deer. Between six and nine oestrous cycles were exhibited by each hind, with no difference in mean cycle length (19.5-19.6 days) between genotypes (P0.10). The overall length of the breeding season was significantly longer for wapiti hybrids (143 days) than for either red deer (130 days) and 14 PD hybrids (132 days, P<0.05). In Trial 2 (1998), adult red deer (n=5), 14 PD hybrids (n=5) and F(1) PD x red deer hybrid (n=5) hinds were maintained together from mid-February (late anoestrus) to early May, in the presence of a fertile red deer stag from 1 April. Thrice-weekly blood sampling yielded plasma progesterone profiles indicative of the onset of the breeding season. Again, there was a significant genotype effect on the mean time to first oestrus (P<0. 05), with F(1) PD hybrids and 14 PD hybrids being 13 and 5 days earlier than red deer. However, conception dates were influenced by the timing of stag joining, and were not significantly different between genotypes. The results indicate genetic effects on reproductive seasonality. However, seasonality observed for PD x red deer hybrids more closely approximated that of red deer than PD deer.

Plasma progesterone and LH concentrations during oestrous synchronization in female fallow deer (Dama dama)

Abstract Fifteen non-lactating adult fallow does were allocated to three treatment regimens (n = 5 does each) designed to synchronize oestrus on or about 28 May (about 4 weeks after the first natural oestrus during autumn). Does assigned to Treatment 1 (natural oestrus) were initially synchronized with intravaginal progesterone CIDRs and the return oestrus was predicted to occur 21 days later on 28 May. Does assigned to Treatment 2 (prostaglandin-induced oestrus) were also initially synchronized with CIDRs and each doe was given an i.m. injection of cloprostenol on Day 13 (26 May) of the subsequent oestrous cycle. Does assigned to Treatment 3 (CIDR-induced oestrus) each received single intravaginal CIDRs for a 14-day period, with CIDR withdrawal occurring on 26 May. Blood samples from all does were collected at 2-h intervals for 70 h from 06:00 h on 26 May (i.e. cloprostenol injection/CIDR withdrawal) to 04:00 h on 29 May inclusive. Plasma samples were analysed for concentrations of progesterone and LH. Only one doe (Treatment 3) was not observed in oestrus during the intensive blood sampling period. Oestrus synchrony was over a 28-h period for Treatment 1, a 12-h period for Treatment 2 (36–48 h from cloprostenol injection) and a 28-h period for Treatment 3 (32–60 h from CIDR withdrawal). Plasma progesterone concentrations of does allocated to Treatment 1 were low ( The doe not observed in oestrus (Treatment 3) exhibited erratic fluctuations in plasma progesterone concentrations throughout the sampling period and failed to exhibit a preovulatory LH surge.

Development of in vitro embryo production systems for red deer (Cervus elaphus) Part 3. In vitro fertilisation using sheep serum as a capacitating agent and the subsequent birth of calves

The following experiments investigated the use of sheep serum (SS) as a capacitating agent for red deer (Cervus elaphus) sperm during in vitro fertilisation. Red deer oocytes were collected at slaughter and matured in vitro for 24h in TCM-199 supplemented with 10% foetal calf serum, 10 microg ml(-1) FSH and LH, and 1microg ml(-1) of oestradiol. Fertilisation medium was IVF-SOF modified to contain 5mM Ca(2+) and no glucose. Experiment 1 investigated the addition of heparin, BSA (8 mg ml(-1)) or 20% SS. All oocytes were penetrated when IVF-SOF was supplemented with SS compared to 10 and 0% penetration when either heparin or BSA was present (P<0.01). However, 43.8% of these oocytes were polyspermic when the medium contained SS. In Experiment 2, the effect of sperm concentration on penetration rates during in vitro fertilisation was investigated. Total sperm penetration and monospermic penetration rates increased with increased sperm concentrations in a log linear manner (P<0.001) and both approached an asymptote at 0.4 x 10(6) sperm ml(-1) with 93.6 and 77% for total and monospermic penetration, respectively. Polyspermic fertilisation also increased with increasing sperm concentrations (P<0.05) but was variable (range 3.5+/-4.2 to 42.3+/-10.6%), especially at the lower sperm concentrations. Experiment 3 investigated the viability of these oocytes after transfer into red deer recipients. Fifteen 2- and 4-cell embryos were transferred into the oviducts of synchronized recipients 28 h post in vitro insemination. An additional fourteen embryos (8-10 cell) were transferred into synchronised recipients after 48 h of in vitro culture in either SOFaaBSA (n=10) or on red deer epithelial oviduct monolayers (n=4). Five (33% 5/15) of the recipients that received 2- and 4-cell embryos were pregnant at Day 45 (verified by ultrasonography) and four recipients subsequently calved. One recipient receiving an embryo cultured in SOFaaBSA was pregnant at Day 45 and subsequently calved. The birth of five normal calves indicate that full developmental competence of red deer oocytes matured and fertilised in vitro can be achieved by the techniques described.

Superovulation of farmed red deer (Cervus elaphus) and fallow deer (Dama dama) : Incidence of ovulation and changes in plasma hormone concentrations during the pre-ovulatory period in relation to ova recovery and fertilisation

Abstract The effects of various gonadotrophin regimens on ovarian responses, endocrine changes and recovery/fertilisation rates of ova in red deer (Cervus elaphus) and fallow deer (Dama dama) were examined. Fifty mature females of each species were treated during their respective breeding seasons with intravaginal progesterone releasing (CIDR) devices for 14 days and one of five doses of ovine follicle stimulating hormone (FSH; 0, 0.25, 0.5, 0.75, 1.0 units). All animals received 200 IU pregnant mare serum gonadotrophin (PMSG) on the 11th day of device insertion and eight equal doses of FSH administered at 12 h intervals starting at administration of PMSG. After removal of CIDR devices, all females were run with fertile males of the same species (10:1 ratio) but also received intravaginal inseminations ((20–30) × 106 motile thawed conspecific spermatozoa per insemination) on four occasions at 12 h intervals starting 24 h after device removal. Four animals in each treatment were blood sampled every 2 days from insertion of CIDR devices until 22 days after device removal and every 2 h for 72 h after device removal. Plasma was analysed for concentrations of luteinising hormone (LH), progesterone, oestradiol-17β and androstenedione. Ova were recovered by flushing the uterus during mid-ventral laparotomy under general anaesthesia 7 days after removal of CIDR devices. The numbers of corpora lutea (CL) and unruptured follicles (over 5 mm) were recorded. Both species exhibited a curvilinear pattern of ovarian response to increasing doses of FSH. The highest numbers of CL were observed following treatment with 0.5 units of FSH. Mean (± SEM) ova recovery rates were 32.7 ± 5.1% for red deer and 30.6 ± 5.1% for fallow deer, with no significant differences between treatment groups. The mean (± SEM) fertilisation rate of ova recovered from red hinds was 50.2 ± 8.2%, and the majority of the embryos were at the morula stage. None of the ova recovered from fallow deer had cleaved. Species differences in pre-ovulatory endocrine changes were apparent, with fallow deer exhibiting more erratic profiles of plasma progesterone and greater elevations in plasma androstenedione than red deer. For both species, concentrations of plasma oestradiol were elevated for multiple ovulating females. We conclude that failure of fertilisation in fallow deer may reflect perturbation of oocyte development,, sperm transport and/or ovulation through excessive secretion of progesterone and oestradiol during the pre-ovulatory period. The unusual patterns of progesterone secretion may be due to either inappropriate follicle development leading to premature luteinisation, or adrenal progesterone as a consequence of stressful treatment regimens. There are indications of marked sensitivity to PMSG in fallow deer.