Synchronisation of oestrus in heifers using steroid (SC5914, SC9880 and SC21009) treatment for 21 days: 1. The effect of treatment on pregnancy rate to artificial insemination

SC5914, SC9880 or SC21009 was administered by daily intramuscular injection of 5.4 mg, 2.4 mg or 0.14 mg respectively. Treatment of 90 cyclical Friesian heifers began on day 4, 11 or 18 of the cycle, and continued for 21 days. Twice daily observations for oestrus were made during treatment and for 28 days thereafter. Heifers in oestrus within 5 days of the end of treatment were inseminated. Pregnancy was diagnosed by palpation at 42 days. 88.9% of the heifers treated were in oestrus within 5 days. Treatment had a highly significant effect on fertility. The pregnancy rate for the SC5914, SC9880 and SC21009 groups were 65%, 72% and 36% respectively. The pregnancy rate of 28 untreated control animals inseminated with semen from the same ejaculate as was used in the treated groups was 93%.     

Synchronisation of oestrus in cattle using a potent progestin (SC21009) and PGF2a

Two experiments are described in which the time relationships of oestrus and ovulation and fertility to artificial insemination in sexually mature Friesian heifers are compared with similar animals treated with PGF₂a alone or in combination with SC21009. It is concluded that neither treatment adversely affects the parameters studied but that the combined treatment will lead to an enhanced oestrous response; and offers a practical method of oestrous synchronisation at farm level.     

Transport of fertilised and unfertilized ova in sows

Transport of fertilised and unfertilized ova was studied in 22 crossbred (Landrace x Yorkshire) multiparous sows. Sows in the inseminated group (I-group, n=11) were inseminated once with 100ml of BTS extended semen from two fertile boars with a total of 10 x 10 (9) spermatozoa during the second oestrus after weaning between 18 and 8h prior to estimated time of ovulation, as estimated from the first oestrus after weaning. All the sows were slaughtered between 36 and 48 h after ovulation in the second oestrus after weaning by stunning and bleeding. After slaughter, the reproductive tract was immediately recovered, the isthmus was divided into three equal segments, and the number of ova was determined in each segment and in the upper third of the uterine horn from the UTJ. There were no significant differences (P>0.05) either in the intervals from ovulation to slaughter (42.3+/-6.2h versus 43.2+/-5.4h) or in the numbers of corpora lutea (CL) (18.2+/-5.5 versus 15.9+/-3.5) between the non-inseminated (N-group) and the inseminated groups (I-group), respectively. Ova recovery rate was 92.5% in the N-group and 82.9% in the I-group (P>0.05). In the I-group, ova had passed 2.2+/-0.3 segments whereas in the N-group, ova had passed 2.6+/-0.3 segments (P=0.38). It can be concluded that there is no difference in the transportation of either fertilised or unfertilized ova in the reproductive tract of pigs.     

Survival of fertilized ova from ewe lambs and adult ewes in the uteri of ewe lambs

The cause of fertility differences between ewe lambs and adult ewes following natural oestrus were studied in Romneys. Insemination was determined by anterior vaginal swabbing. Fertilized ewe-lamb ova were returned to donor animals along with one matched fertilized adult ewe ovum and lambings were recorded. Ewe-lamb ova were less likely (P<0.01) to survive to term compared with adult ewe ova. Insemination failure, fertilization failure and anovular oestrus were minor sources of reproductive wastage.It is concluded that reduced ovum quality appears to be the major cause of the lower fertility in naturally ovulating ewe lambs compared with adult ewes.     

The effectiveness of steers and heifers treated with oestrogen or testosterone to detect oestrus in cattle

Two experiments were carried out to determine the effectiveness of steers and heifers, treated with oestrogen or testosterone, in the detection of oestrus in cattle.In the first experiment 17 steers castrated at birth and 16 castrated at 6 months of age were randomly allocated to three groups and received an 800 mg subcutaneous implant of testosterone, subcutaneous injections of 10 mg oestradiol benzoate per week for 16 weeks or no hormone (controls). In addition, six heifers were injected subcutaneously with 10 mg oestradiol benzoate per week for 16 weeks while six untreated heifers served as controls. Animals were observed in a standardised libido test 2, 4, 8, 16, 20 and 24 weeks after treatment commenced. The time to first mount and the number of mounts per animal responding in the presence of oestrous heifers were recorded. Both steers and heifers treated with oestradiol benzoate were superior at detection of oestrus in cattle than animals treated with testosterone or those receiving no hormone. Oestrogen-treated animals generally detected heifers in oestrus in less than 3 min after introduction and mounted these animals between 20 and 30 times in one hour. This response was consistent throughout the duration of the experiment. There was no effect of age at castration of steers on development of male behaviour.The second experiment determined the rate and degree of development of male behaviour in steers in response to weekly subcutaneous injections of 0, 2, 4, 8 or 16 mg oestradiol benzoate per 250 kg body weight, 250 mg testosterone or 150 mg dihydrotestosterone for a period of 15 weeks. Steers treated with oestradiol benzoate again proved to be more successful than untreated or testosterone-treated steers at consistently detecting and mounting oestrous heifers. The best response was obtained from steers treated with 8 mg/250 kg body weight per week. The practical application of this work is discussed.     

Effects of progesterone or estradiol on uterine tubal transport of ova in the cow

An experiment was designed to determine the effect of progesterone (P) or estradiol benzoate (EB) on uterine tubal transport of ova in the cow. Intramuscular injections of P, EB, or corn oil (C) were administered to heifers 24 hours after the end of estrus. The heifers were euthanatized 60 hours after the end of estrus and the location of the ovum or zygote was determined. Venous serum levels of progesterone and estradiol-17beta were measured by radioimmunoassay. The mean uterine tube (UT) length was 23.9 cm. An ovum or zygote was recovered from 11 of 14 heifers. Serum levels of progesterone and estradiol-17beta were above normal bovine levels following the P and EB treatments, respectively. The mean UT ovum transport rates were 0.42, 0.21 and 0.23 cm/hour in the P, EB and C treatment groups, respectively. The UT ovum transport rate was increased (P<0.05) by the P treatment and EB treatment had no effect (P > 0.05) when compared with the C treatment.     

Effect of nitric oxide synthase inhibitors on ovum transport and oviductal smooth muscle activity in the rat oviduct

The effect of the inhibition of nitric oxide synthase (NOS) on ovum transport and oviductal motility in rats was investigated. Three different NOS inhibitors were injected into the ovarian bursa at oestrus or day 3 of pregnancy. Oviducts and uteri were flushed 24 h later and the presence of ova was recorded. In oestrous and pregnant rats, treatment resulted in accelerated egg transport, as shown by a decrease in the number of ova present in the oviducts. In cyclic rats, intrabursal injection of 1 mg kg-1 of either N-monomethyl-L-arginine (L-NMMA) or N omega nitro-L-arginine methyl ester (L-NAME) elicited a 30% reduction in the number of ova present in the oviducts, whereas in pregnant animals, the same dose of L-NMMA produced a reduction of 40%. Simultaneous administration of the NO donor spermine NONOate (5 mg kg-1) completely reversed the effect of L-NMMA. Tubal motility was assessed by microsphere displacement analysis within the oviduct. Surrogate ova were transferred to the oviductal lumen at oestrus and 24 h later the effect of intraoviductal injection of 1 microgram L-NMMA or vehicle was assessed. The microspheres in the isthmus showed an oscillating motion, and periods in which movement was not detectable. However, L-NMMA treatment produced a 3.6-fold increase in the maximum instant velocities and a significant reduction in the resting periods of the microspheres compared with the control group (P < 0.001). These results provide evidence that NO inhibition increases tubal motility that results in accelerated ovum transport, and indicate that NO could act as a paracrine signal between different layers of the oviductal wall, providing a role for endogenous NO in regulation of tubal function.     

Control of oestrus in cattle using progesterone coils

Four experiments were conducted to determine the effect of length of treatment, stage of cycle at start of treatment and administration of oestradiol benzoate or progesterone at the start of treatment with intravaginal progesterone coils on oestrous response and fertility. In Experiment 1, the number of heifers in oestrus was affected neither by injection of 5 mg oestradiol benzoate alone or with 200 mg progesterone nor by length of treatment. More heifers (P < 0.05) were in oestrus on day 2 after treatment following a 12-day treatment compared to a 9-day treatment.In Experiment 2, heifers between days 17 and 20 of the oestrous cycle received an injection of either 5 mg oestradiol benzoate alone or with 200 mg progesterone at the start of a 9-day treatment with progesterone coils. Neither the number of heifers in oestrus nor the pattern of onset were affected after treatment. In Experiment 3, heifers between days 0 and 3 of the oestrus cycle received progesterone coils for 9, 12 or 14 days. In addition, animals received (i) no further treatment, (ii) a gelatin capsule adhered to the coil containing 10 mg oestradiol benzoate (iii) a gelatin capsule adhered to the coil containing 200 mg progesterone. Following a 9- or 12-day treatment period heifers receiving the coil with the oestrogen capsule had a high oestrous response ( compared to , P < 0.05). When oestrogen was not given, there was a significant linear effect of duration of treatment on the number in oestrus (9 days, ; 12 days ; 14 days, ; P < 0.05).In Experiment 4, post-partum cows were used to compare a 9- and 12-day treatment period and half the animals in each group received either 5 mg oestradiol benzoate and 200 mg progesterone at the start of treatment or a 10 mg gelatin capsule adhered to the coil. The length of treatment affected the number of heifers in oestrus since were in oestrus after a 12-day treatment period compared with after a 9-day period (P < 0.001). There were no significant differences in the number of cows in oestrus after injection of oestrogen and progesterone ( ) or after the use of the gelatin capsule ( ).     

The influence of ovulation number and ovarian dimensions on embryo production in superovulated cattle

Twenty-one cycling Angus heifers and five Holstein cows received a subcutaneous (SC) injection of 50 mg of progesterone (P) in oil for 14 consecutive days. On day 6 of (P) treatment, animals were injected intramuscularly (IM) with 6 mg of estradiol valerate, and on day 13, received an IM injection of 2,000 IU of Pregnant Mare Serum Gonadotropin. Three additional Angus heifers were used as non-hormone treated controls. Seventeen of 21 heifers and 4 of 5 cows (81%) exhibited estrus within 48 to 132 hr following P treatment. Two of the five animals in which estrus was not observed were palpated as pregnant and discarded from the study. Treatment animals showing estrus were randomly assigned either to Group I, animals bred by natural service, or Group II, animals artificially inseminated with two straws of frozen semen at 12-hr intervals for a total of four breedings. Twenty-one animals were slaughtered 2 to 6 days after the onset of estrus, and those animals in which estrus was not detected were slaughtered 10 days after the last P injection. Two of the 24 treated animals had no ovulations. A total of 397 ovulation points ($\text{397}\text{22}$) were counted for a mean ovulation rate of 18 ovulations per animal. One hundred and fifty-six ova were recovered ($\text{156}\text{397}$) for a collection rate of 39%. Group I animals had 44 of 66 (67%) of their ova fertilized while 23 of 71 (32%) of the ova in Group II were fertilized. Nineteen unfertilized eggs were collected from the three animals not observed in estrus. No differences in fertilization rates between the Group I and Group II animals were found. Mean ovarian width, length and weight in the treated animals was measured and found to be 3.5 ± 1.1 cm, 4.8 ± 1.4 cm, and 21.7 ± 21.2 gm, respectively. Ovarian width, length and weight were all positively correlated with the number of ovulations per ovary r=.74, r=.74, and r=.55, respectively. No significant correlation existed between ovarian width (r=.16), lenght (r=.21), or weight (r=.13) when compared to ova recovery rate. This result suggests that ovarian size or weight may not be the limiting factor involved in embryo recovery.     

Effect of progestogen plus estradiol-17beta treatment on superovulatory response in beef cattle

A series of 3 experiments were conducted to evaluate superovulatory response following exogenously controlled follicular wave emergence in cattle. In Experiment 1 the hypothesis was tested that treatments with progestogen plus estradiol-17beta (E-17beta) would result in the emergence of a wave of ovarian follicles that are as responsive to exogenous gonadotropins as those of a spontaneous follicular wave. Beef cows and heifers either received a progestogen ear implant on Day 0 (ovulation) plus 5 mg im E-17beta on Day 1 and were superstimulated on Day 5, or did not receive implants but were superstimulated on Day 8 (expected day of emergence of the second follicular wave). The cattle received 400 mg NIH-FSH-P1 of Folltropin-V, given in a single subcutaneous injection or twice daily as intramuscular injections over 4 d. No significant differences were detected between the 2 groups in the number of corpora lutea (CL), ova/embryos collected, fertilized ova and transferable embryos. In Experiment 2 superstimulatory responses to a single subcutaneous injection of Folltropin-V were compared between heifers in which follicle wave emergence was synchronized with progestogen plus E-17beta at unknown stages of the estrous cycle with those treated following a conventional method of superstimulation at middiestrus. Superstimulation 4 d after E-17beta treatment in heifers with progestogen implants resulted in a similar superovulatory response and higher fertilization rates than those initiated 8 to 12 d after estrus. In Experiment 3 the ovarian response to a single- versus multiple-injection superstimulatory treatment protocol was compared in heifers given progestogen plus E-17beta to induce synchronous wave emergence. The number of CL, ova/embryos collected, fertilized ova and viable embryos were not different between groups. Superstimulatory treatments initiated 4 d after E-17beta treatment of cattle with progestogen implants resulted in comparable ovulatory responses to treatments initiated at the time of spontaneous wave emergence or during middiestrus. Synchronizing wave emergence in a group of randomly cycling cattle obviated the need of estrus detection and synchronization prior to superstimulation.     

Growth and regression of ovarian follicles during the follicular phase of the oestrous cycle in heifers undergoing spontaneous and PGF-2 alpha-induced luteolysis

Ultrasonography was used to monitor the growth, ovulation and regression of individual ovarian follicles greater than or equal to 5 mm during the late luteal and follicular phases of the oestrous cycle in heifers treated with injections of PGF-2 alpha to induce luteolysis and in heifers undergoing spontaneous luteolysis. Six heifers were given a single injection of PGF-2 alpha between Day 12 and 15 of the oestrous cycle and their ovaries were examined daily by transrectal ultrasonography until ovulation occurred. Another group of 5 heifers was examined daily by ultrasound from Day 14 or 15 of the cycle through spontaneous luteolysis and ovulation. Blood samples were taken twice daily from this group and analysed for progesterone to determine when luteolysis occurred. All heifers were checked for oestrous behaviour twice daily. Mean diameters of ovulatory follicles on each of the 3 days before oestrus were not different between PGF-2 alpha-treated and untreated heifers. In both groups there was large variation among heifers in the sizes and growth rates of the ovulatory follicles. At 3 days before oestrus the diameters of ovulatory follicles were between 7.5 and 11 mm in PGF-2 alpha-treated heifers and between 6 and 11.5 mm in untreated heifers. Non-ovulatory follicles decreased in size during the 3 days before oestrus and the number of non-ovulatory follicles within the size ranges of ovulatory follicles decreased. The ovulatory follicle was not consistently the largest follicle on the ovaries until the day of oestrus but was always one of the 2 largest follicles during the 3 days before oestrus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Successful co-culture of 1-4-cell cattle ova to the morula or blastocyst stage

Bovine ova (n = 326) collected at the 1-4-cell stage were cultured in TCM-199 + 10% foetal calf serum with or without oviducal cells. The bovine oviducal cells were collected and seeded either on the day of ovum recovery (BOC-0) or 3 days earlier (BOC-3). In Exp. 1, the effect of age of oviducal cells in co-culture on ovum development was examined. In the BOC-0 and BOC-3 treatments, respectively, 36/46 (78%) and 30/37 (81%) of ova developed to morulae or blastocysts, while no ova developed past the 8-16-cell stage in the absence of oviducal cells. In Exp. 2, the effect of age of oviducal cells and of physical contact between the oviducal cells and ova on ovum development was examined. In the BOC-0 and BOC-3 treatments, respectively, 29/42 (69%) and 23/43 (53%) of the ova developed to morulae or blastocysts, while 1/42 (2%) developed to the morula stage in the absence of oviducal cells. Physical separation of the ova using a microporous membrane inserted between the oviducal cells and the ova did not affect ovum development, with 26/42 (62%) and 22/42 (52%) of ova developing to morulae or blastocysts in the BOC-0 and BOC-3 treatments, respectively. A high proportion of the morulae and blastocysts in Exp. 1 (57/66, 86%) and Exp. 2 (67/100, 67%) were of quality grades 1 or 2, with mean nuclei counts of 85 for morulae and 111 for blastocysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of stage of oestrous cycle on time of oestrus following cloprostenol treatment in the bovine

The first of 2 injections of 0.5 mg cloprostenol (PG1 and PG2) eleven days apart was given to 19 Friesian-Hereford cross heifers between days 8-14 of their cycle (Treatment A) and 16 similar animals between days 0-4 (Treatment B). Oestrus show was monitored by Kamar Heat Mount detectors and vasectomised bulls with chin-ball markers. Blood samples taken at PG1, six days later, at PG2 and four days later were assayed for progesterone to confirm that luteolysis had occurred as expected. Four hourly rectal examinations of the ovaries were carried out from 56-112 hours after PG2 and four hourly blood samples from 36-96 hours after PG2 were collected for FSH and LH assay. Mean time in hours from PG2 to oestrus onset, LH peak and ovulation respectively was 57.4 +/- 2.9, 60.2 +/- 2.0, 91.7 +/- 1.8 for Treatment A and 64.9 +/- 4.1, 68.9 +/- 2.4, 96.7 +/- 1.3 for Treatment B. Treatment A animals showed significantly higher (p<0.01) FSH levels at PG2 than Treatment B. Time from PG2 to LH peak was significantly shorter in animals treated either on days 7 and 8 (p<0.01) or days 15-16 (p<0.05) of their cycle compared with treatment on days 12-14 and it is suggested that these shorter response times correspond to an early and late cycle wave of follicular growth. Secondary FSH peaks some 28 hours after that occurring synchronously with the pre-ovulatory LH peak were observed to be significantly (p<0.01) higher at oestrus associated with the early cycle follicular growth wave as compared with that later in the cycle which may argue a difference in endocrine control of the two periods of follicular maturation.     

Synchronization of estrus in beef heifers with a norgestomet implant and prostaglandin F2alpha

In a 5-year study (1973-1977), 281 cycling beef heifers were treated with a 7-day norgestomet (SC21009) ear implant and an intramuscular injection of prostaglandin F(2alpha) (PGF(2alpha)) at the time of implant removal or 24 hr before implant removal. Percentages of heifers in estrus by 36, 48, 60, 72, and 120 hr after implant removal were 32.4, 52.7, 71.6, 80.1, and 93.2, respectively. Onset of estrus occurred an average of 49.8 +/- 4.7 hr after treatment. Percentages of heifers in estrus 36 hr after treatment were 5.7 and 51.7 for those with a corpus luteum and those without a corpus luteum (or determined regressing by palpation) at implant removal, respectively. When PGF(2alpha) was injected 24 hr before implant removal, 55% of the heifers were in estrus by 36 hr after implant removal compared to 30% when PGF(2alpha) was injected at the time of implant removal; however, by 60 hr after implant removal the difference was 76% vs. 71%. First-service conception rates for synchronized and nonsynchronized heifers were 62.2% and 59.6%, respectively. During 1976 and 1977 heifers were checked for estrus every 4 hr and inseminated 2, 6, 10, 14, 18, 22, 26, or 30 hr after first detected to be in standing estrus. Conception rate was not significantly affected by time of insemination but tended to be higher for heifers bred 26 and 30 hr after first being detected in standing estrus (78.9% and 70.0% vs. average 59.2%). Treatment with a 7-day norgestomet implant plus a single injection of PGF(2alpha) 24 hr before or at implant removal appears to be a practical technique for synchronizing estrus in cycling heifers without affecting conception.     

The use of embryo collection techniques in Holstein heifers: A model to study early embryonic death

Twenty-three cyclic Holstein heifers were purchased for use as embryo donors to study the effect of intrauterine exposure to Haemophilus somnus on the number, quality, and viability of embryos produced. Few problems were encountered using standard superovulation and nonsurgical embryo collection techniques on virgin heifers. Based on three or more ovulations, as determined by palpation per rectum of the ovaries at the time of embryo recovery, 28 of 30 heifers responded to the superovulation regimen. Of 29 nonsurgical recoveries, 27 produced one or more embryos. One hundred and seventy-six embryos and ova were collected from heifers synchronized, superovulated, and flushed 7 to 8 d after insemination.     

Superovulatory response following ablation-induced follicular wave emergence at random stages of the oestrous cycle in cattle

Based on the premise that superovulation in cattle is optimal when superstimulation is initiated at the time of follicular wave emergence, the present study was done in beef heifers to determine if the superovulatory response following a single bolus of gonadotrophin treatment after follicle ablation (induced wave) at random stages of the oestrous cycle is comparable to the same gonadotrophin treatment at mid-dioestrus (spontaneous wave). In Experiment 1, heifers were assigned to nonablation (n = 18) and ablation (n = 20) groups. In nonablated heifers, superstimulatory treatment was given as a single subcutaneous injection (Folltropin-V, 400 mg) at mid-dioestrus to coincide with emergence of the spontaneous follicular wave 8 to 12 days after oestrus. In ablated heifers, the same superstimulatory treatment was given 1 day after ablation of all follicles ≥ 5 mm at random stages of the oestrous cycle to coincide with emergence of the ablation-induced wave. In both the nonablation and ablation groups, PGF_2α (Estrumate, 500 μg) was given 48 h after the superstimulatory treatment and artificial insemination was done 60 and 72 h later. Reproductive tracts were collected at the time of slaughter 6 or 7 days after insemination. Observations made in Experiment 1, indicated that some ablated heifers had only partial luteal regression at the time of insemination, while some others exhibited behavioral oestrus as early as 24 h after PGF_2α treatment. The design was amended in Experiment 2 to address these problems. Heifers were assigned to nonablation (n = 17), ablation-alone (n = 20) or ablation plus progestogen (n = 20) groups. Follicle ablation, superstimulatory treatment, artificial insemination and collection of reproductive tracts were done as in Experiment 1. However, all heifers were given two doses of PGF_2α (500 μg/dose) 48 and 60 h after superstimulatory treatment to ensure complete luteal regression, and heifers in the ablation plus progestogen group received a norgestomet ear implant at the time of follicle ablation to prevent early ovulations. The implant was removed at the time of the second PGF_2α treatment. In Experiments 1 and 2, the means for the ovarian and superovulatory responses were not significantly different between groups. Averaged over the nonablation and all ablation groups for Experiments 1 and 2, the mean number of corpora lutea, fertilized ova and transferable embryos were 22.9 vs 18.6, 7.3 vs 7.8 and 5.4 vs 5.6, respectively. In summary, follicle ablation at random stages of the oestrous cycle followed by a single bolus of gonadotrophin treatment 1 day later resulted in a superovulatory response that was comparable to the same superstimulatory treatment administered around the time of spontaneous wave emergence at mid-dioestrus. The ablation/superstimulation method described herein offers the advantage of initiating superstimulatory treatment forthwith and assuring that treatment is concomitant with wave emergence to achieve an optimal superovulatory response. Moreover, the full extent of the oestrous cycle is available for superstimulation and the need for detecting oestrus or ovulation and waiting 8 to 12 days to initiate treatment is eliminated.     

Ovulatory responses and plasma luteinizing hormone concentrations in dairy heifers after treatment with exogenous progesterone and estradiol benzoate

The objectives of this experiment were to determine the effects of 0.5 mg estradiol benzoate, administered intramuscularly 24 h after removal of CIDR-B progesterone containing intravaginal devices, on the time to estrus, ovulation and peak LH concentration in dairy heifers. Ovulatory responses and plasma LH concentrations were examined using 14 Friesian dairy heifers in 2 separate treatment periods. All heifers received a CIDR-B progesterone-containing intravaginal device with an attached 10-mg estradiol benzoate capsule for 12 d. Within each period, 24 h after CIDR-B removal, 7 heifers received an intramuscular injection of 0.5 mg estradiol benzoate while the remaining 7 heifers received an intramuscular injection of a placebo. Blood samples for LH assay were collected at 0, 6 and 12 h, and then every 4 h for 60 h after estradiol injection. Detection of estrus was conducted at 4-h intervals, and ultrasonographical examination to detect ovulation was conducted every 8 h for 88 h after removal of the CIDR-B device. Treatment with estradiol benzoate tended to reduce the time from device removal to the LH peak in Period 1 (median time to LH peak 40.1 vs 63.9 h; P = 6.07). In Period 2, treatment with estradiol had no significant effect on the time to the LH peak, standing estrus or ovulation. We hypothesize that the period effect was due to the stage of cycle at the time of treatment. For heifers treated in Period 1, the stage of cycle was random. However, because of the prior synchronization of estrus, which was implicit in the experimental design, heifers in Period 2 tended to be in late diestrus. The administration of estradiol benzoate after treatment with exogenous progesterone appears to overcome the variability in timing of LH peaks typically occurring in a herd of synchronized heifers due to different stages of follicular development.     

Synchronization of Oestrus in Heifers with Norethisterone

Sixty-five heifers in different stages of the oestrus cycle were fed norethisterone once daily for 17 days at doses of 0.2, 0.6 and 1.0 mg/kg body weight. During treatment swollen vulva, mucus discharge, open and reddened portio and udder enlargement were noted. Norethisterone was effective in suppressing oestrus and ovulation at doses of 0.6 and 1.0 mg/kg. Extremely good heat-synchronization followed the treatment and 98 % of the heifers came on heat within a two-day period. Fifty-five % conceived after the first insemination. In the group receiving 0.2 mg norethisterone per kg, three out of 13 heifers showed psychic heat during treatment. Poor heat synchronization and low conception rate were obtained in this group. A possible effect of cycle stage on heat synchronization was noted only in the 0.2 mg group where a better result was obtained when treatment began in the follicular phase.     

Persistence of human mitochondrial DNA throughout the development to the blastocyst of mouse zygotes microinjected with human mitochondria

The conditions for transfer of human mitochondria into fertilised mouse ova were elaborated. Species-specific primers were designed to discriminate human mitochondrial DNA (mtDNA) and the endogenous mtDNA in the preimplantation embryos. Human mitochondria isolated from the HepG2 cell line were microinjected into murine zygotes, and the latter cultured for 96 h to the blastocyst stage. The polymerase chain reaction allowed the detection of human mtDNA at every stage of embryo cleavage. In some cases a clear disparity in distribution of human mtDNA among blastomeres was evident.     

The effect of time of intrauterine insemination on the development and viability of embryos collected from superovulated ewes

Eighteen Border Leicester x Scottish Blackface ewes, primed with 300 mg progesterone (12 d) and superovulated with decreasing doses (6, 5, 3 and 2 mg) of porcine FSH, were inseminated with fresh semen, using laparoscopic intrauterine procedures at 48 (Group E) or 60 h (Group L) after exogenous progesterone removal. Five days after insemination, embryos were collected and classified on the basis of their morphological development. During the subsequent 3 d of in vitro culture (38.5 degrees C; 5% CO2) the embryos were evaluated at 24-h intervals. After 72 h, the embryos were individually fixed (24 h) and stained with aceto-orcein and the nuclei were then counted to provide an objective index of cell proliferation and development. Mean (+/-SEM) ovulation rates for the 2 groups (9.2+/-1.5 and 7.1+/-1.2, respectively) and the corresponding percentages (53 vs 59) of embryos collected by laparoscopy were unaffected by insemination time. All donors yielded fertilized ova, but whereas all Group-E donors yielded 1 or more viable embryos (i.e., >32 cells), only 5 Group-L ewes yielded viable embryos (P<0.10). At collection, the percentages of embryos at the morula stage of development were 98 (Group E n = 44) and 39 (Group L n = 38; P<0.001). Few of the remaining ova (Group E = 0% Group L = 8%) were at the 1-cell stage of development when collected, indicating that retarded development post fertilization, not fertilization failure, was the principal consequence of delayed insemination. The percentages of embryos that continued to develop during in vitro culture were 91 and 37 for Groups E and L, respectively (P<0.001), and all of these reached the blastocyst stage. Of these blastocysts, 75 and 50% in Groups E and L hatched in vitro (P<0.10), with mean (+/-SEM) nuclei counts of 148+/-22.7 and 76+/-13.8 (P<0.02), respectively. In conclusion, while delayed intrauterine insemination did not affect the efficiency of ovum collection, it caused a major reduction in the yield of embryos that were capable of developing during in vitro culture. However, fertilization failure accounted for only 13% of the loss in viability following late insemination.