Use of stored goat semen and factors influencing fertility after insemination

into the cells creates membrane damage. As well, lipid reorganisation because of Ž . thermal changes alters membrane sensitivity to various stresses. Watson 1995 proposed some intriguing theories on cryoinjury with significant consequences for future studies. He assumed that either sperm recovering after freeze-thawing are a subpopulation of spermatozoa, which are viable but relatively infertile, or that freezing generally modifies cell membranes, the survivors being affected in some deleterious way. The destabilisa- tion of membranes leads to premature acrosome reactions, shortens the life span and reduces fertility. Thus, frozen-thawed spermatozoa are partially capacitated and capable of achieving fertilisation over a short period of time. This concept fits well with the Ž practical methods of insemination developed for the use frozen-thawed semen see . later . The above theories represent a change in direction of research for semen storage. Future work may focus on modifying membrane structures before freeze-thawing to render them resistant to cryoinjury. A reversible membrane change that increased resistance would undoubtedly lead to higher fertility.

4. Use of stored goat semen and factors influencing fertility after insemination

Chilled-stored and frozen-thawed semen is used for insemination in natural, synchro- nised or induced oestrus in which several factors can influence the fertility. 4.1. Natural oestrus Successful AI at natural oestrus depends in the first instance on an efficient method of oestrous detection. Sexual receptivity of the female is evidenced by mounting by the male, or more particularly by the postural immobilisation of the female in response to mounting by the male. Different practical methods of oestrus detection are used, including entire males provided with an apron, vasectomized males or androgenized females fitted with a harness and marking crayon. These ‘teasers’ may be run with the flock and marked does removed once or twice per day, or they may be taken to the females at intervals and their behaviour observed directly. In order to control the time of oestrus in the normal breeding season, simple methods Ž . make use of the ovulation inducing effect of bucks ‘buck effect’ and oestrus females Ž . Ž ‘female effect’ to synchronise oestrus and ovulation Chemineau, 1987; Restall, 1988, . 1992 . 4.2. Synchronised and induced oestrus In large commercial herds economic considerations necessitate the synchronisation of oestrus in females which are cycling naturally, or its induction during the nonbreeding season, with insemination performed at a fixed time either in relation to the beginning of oestrus or the end of a hormonal treatment, PMSG. The hormonal treatment generally used to induce oestrus and ovulation is based on combinations of progesterone or a progestagen treatment followed by eCG, and may include treatment with commercial prostaglandin analogues. Hormonal treatments based Ž on a double prostaglandin injection in cycling females have also been used Bretzlaff et . al., 1981 . A synchronisation treatment used on a large scale in France for dairy goats consists of: insertion of a vaginal sponge impregnated with a synthetic analogue of Ž . progesterone 45 mg fluorogestone acetate: FGA for 11 days, intramuscular injection of Ž . eCG 400 to 600 IU according to the milk production and season of treatment and 50 mg of cloprostenol, both administered 48 h before sponge removal. Treated goats are inseminated once with 100 million frozen-thawed spermatozoa, at 43–45 h after sponge Ž . removal Corteel et al. 1988; Corteel and Leboeuf, 1990 . The fertility rate in dairy goats Ž . after one insemination was 60–65 Boue and Sigwald, 1995 . In the Australian ´ Ž . cashmere goat, Ritar et al., 1990b indicated that controlled internal drug release Ž . CIDR devices containing progesterone were equally effective as intravaginal sponges for the control of ovulation, when combined with an injection of eCG. Greyling and Van Ž . Nickerk 1991 , in the noncyclic Boer goat, have successfully used vaginal sponges Ž . impregnated with medroxy-progesterone acetate MAP for 14 days. Photoperiodic treatments have been used to induce oestrous cyclicity in goats outside the breeding season. Such treatments involve application of extra artificial light, with a fixed dawn and a nocturnal lighting, for 2 months, before administration of a melatonin Ž . implant for 40–50 days Chemineau et al., 1996 . After this treatment, AI can be performed at the natural oestrus. Association of photoperiodic and hormonal treatments, synchronisation of oestrus and AI is under current investigation. Ž . When oestrus was induced by treatments with progestagen FGA sponges for 18–21 days and eCG administration 48 h before sponge removal, the kidding rates after two intracervical inseminations with frozen-thawed spermatozoa were dependent upon motil- Ž . Ž . ity of spermatozoa Corteel et al., 1988 Table 2 , and probably involved impaired sperm transport through the cervix due to prolonged administration of FGA, as it has Ž . been shown in sheep Quinlivan and Robinson, 1969 . When FGA treatment was shortened to 11 days, followed by eCG and Cloprostenol administration, the kidding Ž . rate, measured over several years, was increased from 56.7 n s 6210 to 61.1 Ž . n s 6970; Corteel et al., 1988 . After deposition of the semen into the cervix, fertility Table 2 Effect of duration of progestagen treatment on the kidding rate after insemination into the cervix a with Ž . frozen-thawed semen, adapted from Corteel et al., 1988 Ž . Duration of FGA treatment days Motility scores F1.0 1.5 G 2 Ž . Ž . Ž . 18–21 46.3 1222 51.6 533 57.9 1344 Ž . Ž . Ž . 11 53.9 349 60.9 220 61.0 264 Ž . Number of females; a Females inseminated twice with 150–200 million spermatozoa. P - 0.05. P - 0.01. Table 3 The effect of site of deposition of frozen-thawed semen in kidding rate in adult goats after progestagen Ž . treatment adapted from Corteel et al., 1988 Ž . Duration of FGA treatment days Site of semen deposition Cervix Uterus Ž . Ž . 18–21 51.7 3392 62.6 2848 Ž . Ž . 11 59.3 3970 64.3 2156 Ž . Number of females inseminated. P - 0.01. Ž . was influenced by sperm motility Table 2 . In females hormonally treated for 18–21 days the fertility was lower after semen deposition into the cervix than into the uterus Ž . Table 3 . Similar results have been obtained in Angora goats, with fertility increasing with the depth of insemination and little difference between fresh-diluted or frozen- Ž . Ž . thawed semen after intrauterine insemination Table 4 Ritar and Salamon, 1983 . 4.2.1. Antibodies against eCG PMSG The techniques using FGA reCG treatments with AI at a predetermined time after sponge removal can result in variable fertility rates due to variation in the time of oestrus and ovulation. The fertility of goats which came into oestrus more than 30 h after sponge removal was significantly lower than those which were first observed in Ž . oestrus 24 or 30 h after sponge removal 33 vs. 65 respectively, Baril et al., 1993 . The distribution of the time of onset of oestrus after sponge removal was affected by the Ž . number of hormonal treatments previously received by the females Baril et al., 1996 . The delay in the onset of oestrus behavior was associated with a delay in preovulatory Ž . Ž surge of LH Maurel et al., 1992 and a delay in the time of ovulation Leboeuf et al., . 1996 . In another French survey, eCG binding in plasma before the onset of treatment was found to be significantly lower in flocks never treated, compared to flocks with a history of eCG treatments, and was not dependent on the age of females. eCG binding was higher in females that had previously received two to five treatments compared to Ž females that had no or only one treatment. Fertility decreased 51 vs. 66, 166 vs. . Ž . 353 females , when eCG binding was higher than 5. Baril et al., 1996 . Table 4 Ž . Effect of depth of insemination with fresh and frozen-thawed semen on kidding rate in Angora goats Ž . adapted from Ritar and Salamon, 1983 Depth of cervical insemination Fresh diluted semen Frozen-thawed semen Ž . Ž . Up to 1 cm 40.9 88 27.0 63 Ž . Ž . 1.0 to 3.0 cm 53.3 127 45.9 85 Ž . Ž . Into the uterus 69.1 81 68.6 102 Ž . Number of females. 4.3. Other factors influencing fertility 4.3.1. Characteristics of indiÕidual animal Irrespective of the process used for freezing, differences have been observed between males regarding the freezability and fertility of semen, so they could be classified as ‘good freezers’ or ‘bad freezers’. This variability is relatively independent of prior semen quality, and the semen of certain individuals consistently freezes with less Ž . cryoinjury than that of others Corteel et al.,1987 . Spermatozoa acquire cold shock Ž . sensitivity as they traverse the epididymal tubules Watson, 1981 , and this is believed to be related to changes in membrane lipids during the epididymal transit. Differences in Ž . either ejaculation frequency, previously indicated by Boue and Corteel 1992 , or in ´ epididymal transit time and sperm mixing in the epididymis may provide a potential mechanism for variability in response to subsequent temperature fluctuation, explaining Ž why ejaculates within individuals can vary in their responses to freeze-thawing Watson, . 1995 . 4.3.2. Season of semen production Reports on the freezability and fertility of semen collected in the breeding and nonbreeding season are contradictory. Some investigators found that freezability of Ž . Ž semen Muhuyi et al., 1992 and fertility of frozen-thawed spermatozoa Corteel et al., . 1978 was better when collected in the breeding than in the nonbreeding season. Other Ž . workers Peskovatskov et al., 1974; Summermatter and Flukiger 1982 observed no Ž . seasonal difference in fertility. According to Aamdal 1982 in Norway for frozen storage the semen is collected before the breeding season Ž . Corteel et al. 1980 have shown that the kidding rate of dairy goats inseminated with frozen-thawed spermatozoa after hormonal induction of oestrus and ovulation, was Ž . significantly lower with semen obtained outside the breeding season 53, n s 549 Ž . than with semen processed in the breeding season 61, n s 998 . When semen stored at 48C for less than 12 h was used for insemination of dairy goats outside the breeding season, the kidding rate was significantly lower than in goats inseminated with frozen- Ž thawed semen obtained during the previous breeding season, 52.1, n s 4505, and Ž . 56.7, n s 6240, respectively Corteel et al., 1988 . These observation suggest that the semen of valuable sires intended for long-term storage should be collected and pro- cessed during the normal breeding season. There has been little research to date aiming to overcome the seasonal differences in semen quality. 4.3.3. Physiological condition of females The physiological condition of the doe varies with parity, season, level of production and age, and all these factors have been found to influence the success of hormonal treatments to induce synchronisation of oestrus and ovulation for the subsequent AI. Fertility after oestrus synchronisation and AI is low during postpartum and lactational anoestrus. The latter increases significantly with time elapsed from parturition and fertility is only fully restored if the hormonal treatment is delayed until 4 full months Ž . after parturition Corteel, 1975b . The time between kidding and re-breeding affects the fertility of goats. Increasing the length of time between weaning and laparoscopic insemination with frozen-thawed semen from 3 weeks to 3 months improved the Ž . proportion of Cashmere does kidding from 47 to 65 Ritar et al., 1989 . In the high latitudes, the percentage of anoestrous females is highest in the middle of the nonbreeding season, well in advance of the normal restoration of cyclicity. To minimize this handicap in large-scale commercial AI programs in France, higher doses Ž of eCG have been administered before 15 June midsummer, 500 IU instead of 400 IU . after June . This has resulted in small fertility differences between treatments in the Ž . breeding and nonbreeding seasons Leboeuf, 1989 . To reduce the depressive effect of high levels of milk production on fertility, Leboeuf Ž . 1989 injected 600 IU eCG before 15 June and 500 IU of eCG after 15 of June in goats producing higher than 3.5 kg of milk per day during the month preceding the hormonal treatment. The increase in the dose of eCG tended to improve the kidding rate of very Ž . Ž . high milkers: 47.8 n s 138 vs. 43.9 n s 139 . 4.3.4. Age and parity After hormonal induction of oestrus, nulliparous goats were less fertile than primi- Ž . parous or multiparous goats when inseminated intracervically Leboeuf, 1989 . Ritar and Ž . Ball 1991 indicated that maiden goats should be inseminated laparoscopically rather than cervically because of the difficulty in opening the vagina with a speculum. Little difference in fertility was observed between 18-month-old maiden does and mature does Ž . when they were inseminated laparoscopically Ritar and Ball, 1993 . 4.3.5. Time of insemination 4.3.5.1. Natural oestrus. Successful fertilisation depends on the time of insemination Ž . relative to ovulation. Dauzier 1966 showed that the fertility of goats inseminated with Ž fresh semen at different time intervals from the onset of natural oestrus observed by the . Ž . farmer was highest after insemination at the beginning of the oestrus period Table 5 . Ž This is in accordance with the known time of ovulation Harrison, 1948; Lopyrin, 1953; . Gonzalez-Stagnaro et al., 1984; Rao and Bhattacharya, 1980 , duration of sperm Ž . transport in the female genital tract Ajello, 1958; Lopyrin, 1953 and time of survival of Ž . male and female gametes Lopyrin, 1953 . Females in natural oestrus are generally inseminated, after once daily detection, at the first, or highly fertile, stage of the oestrus period. Table 5 Ž . Ž Effects of method and time of insemination with fresh-diluted semen on kidding rate adapted from . Dauzier, 1966 Time of insemination after oestrus detection Method of insemination Ž . Cervical and intrauterine via the cervix Intravaginal Ž . Ž . First 12 h 74.8 298 66.9 444 Ž . Ž . 12–24 h 66.2 320 60.7 326 Ž . Ž . Later than 24 h 51.0 100 44.6 148 Ž . Number of females inseminated. 4.3.5.2. Hormonally synchronized oestrus. When intravaginal progestagen treatment is used out of breeding season, administration of eCG is imperative to ensure ovulation, and insemination should be carried out in relation to the time of sponge removal. In Ž . Ž . French protocols, cervical inseminations are performed 43 h Alpine or 45 h Saanen Ž . after sponge removal Corteel and Leboeuf, 1990 . In programmes for fibre-producing goats, cervical inseminations occurred 48–59 h after FGA sponge removal and eCG Ž . injection Ritar and Salamon, 1983 , 40–48 h after sponge removal when eCG was Ž . injected 48 h before removal Ritar et al., 1989 , and 10–15 h earlier if CIDRs rather Ž . than sponges were used Ritar et al., 1990b . 4.3.6. Volume of inseminate and number of spermatozoa The total number of spermatozoa inseminated per female is one of the main factors affecting fertility, and it is necessary to know the threshold at which the required fertility is reached for a given breed with females synchronised by particular methods for various conditions of semen storage. Ž . Corteel et al., 1988 reported that fertility after one insemination with 200 million frozen-thawed spermatozoa in a 0.5-ml straw was similar to that obtained after two inseminations with 2 = 200 million spermatozoa in hormonally synchronised dairy Ž . goats. Further work Corteel and Leboeuf, 1990 demonstrated that acceptable fertility Ž . around 60 may be obtained with a reduction in the number of spermatozoa inseminated from 200 to 100 million in Alpine goats and to 60 million in Saanen goats, with a concomitant reduction in inseminate volume from 0.5 to 0.2 ml. Ritar and Ball Ž . 1993 found that one and two inseminations were equally effective in natural or hormonally synchronised oestrus in Angora goats, provided the dose of spermatozoa exceeded 120 million. 4.3.7. Site of semen deposition In goats in which oestrus and ovulation was induced, laporoscopic intrauterine insemination resulted in improved fertility rates and allowed a reduction in numbers of Ž frozen-thawed spermatozoa inseminated 5–20 million motile spermatozoa; Ritar et al;, Table 6 Percentage of adult cashmere females pregnant after cervical and laparoscopic insemination with frozen-thawed Ž . semen adapted from Ritar et al., 1990b Main effect Cervical Laparoscopic IntraÕaginal treatment Ž . Ž . CIDR 40.7 204 64.5 290 Ž . Ž . Sponge 37.4 198 62.7 279 a Time of insemination after treatment Ž . Ž . Before ovulation 44.0 200 69.6 283 Ž . Ž . After ovulation 34.2 202 56.7 286 Ž . Number of females. a Ž . Does were inseminated cervically either before 45 h after CIDR removal; 55 h after sponge removal or Ž . after 55 h after CIDR removal; 65 h after sponge removal the estimated time of ovulation. Table 7 Percentage of cleaved eggs and number of good embryos related to the interval between the onset of oestrus Ž . Ž and the insemination time adapted from Vallet and Baril, 1990 laporoscopic insemination with 100 million . frozen-thawed spermatozoa Ž . Interval between onset of oestrus and insemination h - 12 14–18 20–24 26–30 32 of cleaved eggs 22.0 57.2 78.3 57.2 29.6 Number of embryos per goat treated 0.6 3.0 6.4 4.4 3.2 Number of females 8 22 61 33 11 . 1990b . Comparisons between laparoscopic and cervical inseminations in dairy goats Ž have shown significant differences in fertility rates 62.6 vs. 49.3; P - 0.05, . respectively,Vallet et al., 1992 , regardless of the low numbers of total spermatozoa deposited: 10 million sperm, 61.5 vs. 20 million sperm, 63.7. Fertility was also better after laparoscopic than cervical insemination with frozen-thawed semen in Aus- Ž . Ž tralian feral goats Moore et al., 1988 and Australian cashmere goats Ritar et al., . 1990b; Table 6 . 4.3.8. SuperoÕulation The transport and survival of spermatozoa in the female tract are disrupted after Ž progestagen treatment associated with the hormonal induction of superovulation Evans . and Armstrong, 1984 . The detrimental effect of the altered uterine environment on spermatozoa was not reduced by increasing the number of inseminations and the number Ž . of spermatozoa per female 2 vs. 3 million , and only 48.6 of cleaved eggs were Ž obtained after cervical insemination with frozen-thawed spermatozoa Vallet et al., . 1991 . However, an increased percentage of cleaved eggs resulted from laparoscopic insemination with 100 million frozen-thawed spermatozoa, provided the insemination Ž . was performed 20–24 h after the onset of oestrus Vallet and Baril, 1990, Table 7 .

5. Conclusion and perspectives