Reciprocal embryo transfer between repeat breeder and virgin heifers--an experimental model

The aim of the study was to evaluate the importance of mother-concaptus relationships for the elevated embryonic loss in repeat breeder heifers. Embryos were collected by non-surgical technique, classified and transferred surgically or non-surgically to synchronized, inseminated recipients. The embryos were transferred to the uterinehorn contralateral to the corpus luteum. The embryos were transferred from repeat breeder heifers (RBH) to virgin heifers (VH) or from VH to RBH. After slaughter 4 weeks after transfer there was no difference in emhryonic survival between heifer categories following transfer or insemination. In some animals degenerated foetal membranes were found in the nonpregnant horn. The study indicates embryonic morphology rather than the category of donor or recipient as influencing the embryonic survival rate.Key words: embryo, embryonic death, embryo transfer, repeat breeder     

Successful,repeated non-surgical collection of blastocysts from virgin and repeat breeder heifers

A total of 55 non-surgical collections, 7 days after spontaneous heat, was made in 19 heifers. Recovery rate was 57% (17/30) from 10 repeat breeder heifers and 72% (18/25) from 9 virgin heifers, this difference was not significant. Fertilization rate in both groups was 94%. The eggs were always recovered in the first 100 ml of flushing medium. Mean lengths of oestrous cycle after non-surgical collection were, respectively, 16.9 and 18.1 days in repeat breeder and virgin heifers. This difference was not significant. However, the cycle in which the first collection was made was significantly shorter (P<.005) than the cycle immediately before the collections. Only 61% of possible eggs were non-surgically collected from 4 superovulated repeat breeder cows and no further eggs were found after slaughter, one to two hours later.     

Concentrations of luteinising hormone and progesterone in pregnant and non-pregnant heifers

An experiment was conducted to determine if concentrations of luteinising hormone or progesterone were different in pregnant or non-pregnant heifers for seven days before and 20 days after a successful or non-successful insemination. Heifers with an oestrous cycle length of 18 to 24 days only were used and they were bled at 08.00, 16.00 and 24.00 each day for seven days before and for 20 days after insemination with thawed semen (treatment 1) or semen diluent (treatment 2). Animals allocated to treatment 3 had the embryo nonsurgically flushed from the uterus at days 10 to 12 while animals allocated to treatment 4 were inseminated with semen diluent and then had a viable embryo transferred to the uterus between days 10 and 12. All animals were slaughtered between 19 and 21 days after insemination and pregnancy rate determined. There were no differences in basal luteinising hormone levels between treatments. Blood concentrations of progesterone were not different before insemination and for 16 days after insemination for pregnant (11 out of 15) and non-pregnant heifers (14) allocated to treatments 1 and 2. Between days 17 and 20, progesterone concentrations declined in non-pregnant heifers. Transfer of an embryo to non-pregnant heifers on day 10 to 12, did not affect progesterone concentrations, but non-surgical flushing of the embryo caused a decline in blood concentrations of progesterone. It was concluded that basal blood concentrations of luteinising hormone and progesterone, in samples taken three times daily were not different in pregnant or non-pregnant heifers before and for 16 days after insemination.     

Karyotypes and morphological and histological alterations of the genital tracts of repeat breeder heifers with known breeding history

Forty-two repeat breeder heifers (RBH) and 27 virgin heifers (VH) were used in the study. The breeding history and the occurrence of chromosomal aberrations were investigated in the RBH. The genital tracts of the RBH and the VH were investigated at slaughter 3 to 35 days after artificial insemination and/or embryo transfer for the presence of morphological and histological changes. According to AI data the mean number of inseminations per RBH in the herd of origin was 4.9 and 110 interservice intervals out of 141 calculated were normal (17–24 days) or twice normal (34–48 days). Two RBH out of 28 investigated demonstrated the 1/29 translocation. The post mortem examination revealed corpus lutea and follicles normal for the stage of the cycle in all animals. In three RBH abnormalities of the uterus or cervix were found. A higher incidence of cystic glands and of focal accumulation of erythrocytes and lymphocytes in the endometrium was found in the RBH than in the VH (P ≤ 0.05). The relation between the embryonic survival and the histological alterations of the uterus was not significant.It is suggested that errors in heat detection, abnormal karyotypes and morphological defects of the genital tract play a minor role in the repeat breeding complex in heifers.     

Intrauterine Neospora caninum inoculation of heifers

Here, we studied the potential of Neospora caninum tachyzoites to infect heifers when administered in utero by artificial insemination via contaminated semen. Eighteen primiparous cyclic heifers were hormonally synchronized and artificially inseminated. Nine of them, which were inseminated with semen containing 10(7) live N. caninum NC-1 isolate-tachyzoites, reacted with seroconversion and a specific IFN-gamma response. Moreover, N. caninum DNA was demonstrated by a nested-PCR in the blood of all nine heifers and in brain, lungs, liver and uterine horn of several of them. In contrast, nine heifers inseminated with tachyzoite-free semen developed no antibody or IFN-gamma responses, and no parasite DNA was detected in blood or organs. At necropsy, viable embryos were detected in one and six of the infected and non-infected heifers, respectively. No specific Neospora DNA was detected in any of the embryos. This study provides evidence that intrauterine inoculation via contaminated semen cause N. caninum infection in cattle.     

The Morphology of 16 and 17 Day Old Bovine Blastocysts from Virgin and Repeat Breeder Heifers

The morphology of 16–17 days old embryos from virgin heifers (VH) and repeat breeder heifers (RBH) was compared using light and electron microscopy. In addition some embryos transferred from one heifer category to the other were studied. Embryos from VH were elongated blastocysts and the oval embryonic disc had three germ layers. The ectoderm was stratified and many mitoses were seen. The endoderm lining the blastocoelic cavity consisted of almost squamous cells conjoined by tight junctions. Between the ectoderm and the endoderm the mesoderm had developed and expanded laterally and the coelom had formed. The trophoblastic cells adjacent do the embryonic disc were cylindrical, whereas those more peripheral located were cuboidal. The trophoblastic cells were conjoined by tight junctions and they had numerous long microvilli on their peripheral surface. Except in the embryonic disc region, the endodermal cells had filopodial processes towards the trophoblast. The embryos from RBH varied in appearance. One was similar to those from VH whereas the others were, more or less retarded, without formation of mesoderm. The smaller one consisted ot trophoblastic cells only. The transferred embryos (representing surviving embryos: 2 out of 9 in VH-RBH and 5 of 6 in RBH-VH) had a morphology similar to that of VH blastocytes two though, appeared somewhat retarded. It is suggested that the retarded embryos lack the ability to complete embryonic development and that the uterine environment of RBH is not favourable to sustain normal embryonic development.     

Early diagnosis of pregnancy in sows by ultrasound evaluation of embryo development and uterine echotexture

Two operators attempted to detect pregnancy ultrasonographically in 196 sows daily from 15 to 25 days after insemination; 20 unbred sows were also investigated. The probe was applied transcutaneously on the right abdominal wall near the last three mammary glands. During each examination, the embryos were visualised and their transverse and longitudinal dimensions were measured. Pregnancy was confirmed by an ultrasonographic detection of embryos five days after the first ultrasound diagnosis and finally 30 to 32 days after insemination. The accuracy of diagnosis was less than 83 per cent on days 15, 16 and 17 but improved to more than 90 per cent from day 18 onwards. The uterine echotexture was studied in seven sows at oestrus and 15, 17, 19, 21, 23 and 25 days after insemination. The echotexture was more homogeneous from days 15 to 25 after insemination than at oestrus.     

Non-surgical transfer of bovine embryos

Bovine embryos obtained from donors six to nine days after oestrus were transferred non-surgically at a rate of one per recipient using a sterile insemination instrument, protected from contamination by the vagina with a plastic sheath. The percentage of recipients pregnant increased with the age of embryo transferred and for day 6 and 7 embryos was 33% compared to 58% for day 9 and 8 embryos. This difference approached statistical significance. Bacterial contamination of the instrument on withdrawal after transfer was not related to the success or failure of pregnancy. Maintenance of pregnancy to term and calving appeared to be normal. It is suggested that this method could be used for the routine transfer of eight and nine day embryos.     

Relationships among estrous behavior, superovulatory response and grade 1 embryo sex ratio in superovulated Holstein heifers

In this study, we first attempted to determine whether the timing of artificial insemination affects the sex ratio of seven-day-old embryos in superovulated Holstein heifers. The superovulatory treatment consisted of eight decreasing doses of FSH for 4 days and 2 doses of PGF(2alpha) given with the last two doses of FSH. The superovulated heifers were given a GnRH analogue 48 h after the first PGF(2alpha) treatment and were artificially inseminated 48 h (n=10) or 56 h (n=8) after the first PGF(2alpha) treatment. There were no significant differences in the percentages of unfertilized ova and transferable embryos (grades 1 to 3) between the two groups. The proportions of female grade 1 embryos did not significantly differ from the expected ratio of 50:50 (49.3% at 48 h and 52.5% at 56 h). We then compared the estrous behavior and superovulatory responses of the heifers with a proportion of female embryos of 50% or less (n=7, Low group) to those of the heifers with a proportion of female embryos of more than 50% (n=9, High group). The Low group had a longer duration of estrus and a higher superovulatory response than the High group. These findings offer little encouragement for prediction of the population of female embryos collected from superovulated heifers. Further studies are necessary to evaluate to what degree maternal hormone levels are related to estrus duration and sex ratio.     

受体绵羊人工授精后移植胚胎的试验

试验通过使用人工授精后绵羊作为受体进行胚胎移植,以寻找一种更加有效的方法提高绵羊胚胎移植的经济效益。试验中使用FSH对10只无角道赛特绵羊进行超数排卵处理,同时对60只受体小尾寒羊进行同期发情。供体羊在发情配种后4.5～5.0d从子宫角收集胚胎。同时,将胚胎移植到同期发情并进行人工授精的受体羊子宫内。总共有57枚可用胚移植给44只受体小尾寒羊,32只怀孕到分娩,共产下羔羊51只(无角道赛特羔28只,道赛特与小尾寒羊杂种羔23只)。此外,经人工授精但未进行手术移植的7只小尾寒羊产下15只杂种羔羊。移胚植受体妊娠率72.7%(32/44),移胚受体繁殖率118%(51/44),受体利用率88.3%(53/60)。移胚受体总妊娠率和受体利用率均显著高于常规ET组(P<0.01)。与常规胚胎移植相比,受体羊人工授精后移植胚胎不仅提高了无角道赛特母羊的繁殖率,而且提高了受体羊的利用率。     

Megestrolacetat applied for oestrusinduction and oestrussynchronization in heifers (author's transl)]

Forty-seven heifers with the diagnosis anoestrus were treated with 15 mg to 60 mg megestrolacetat (Niagestin "NOVO") per os for 10 days. Thirty heifers showed oestrus within 10 days after treatment and 56% of these conceived after first insemination. There was no significant difference in fertility after different doses of megestrolacetat. Ten cycling heifers were synchronized with 50 mg megestrolacetat per os for 14 days. On the first day of treatment 5 mg oestradiolvalerat was given intramuscularily. All heifers came into heat and were inseminated 2--4 days after treatment. Fifty per cent were pregnant 55 days after insemination. Dosage of megestrolacetat for cows and heifers are discussed.     

Plasma levels of prostaglandin F2 alpha metabolite and progesterone in repeat breeder heifers.

A detailed clinical-endocrine investigation was performed in 6 repeat breeder heifers (RBH) with the aim being to ascertain whether endocrine asynchronism exists at luteal regression and during early pregnancy. The heifers were first studied during an open cycle and then after insemination when 3 heifers became pregnant. Circulating plasma levels of PGF2 alpha metabolite were measured every 2nd h, while progesterone (P4) levels were measured every 6th h. The oestrous period and intervals between the onset of oestrus and ovulation were relatively longer, compared with what is normally seen in heifers. Plasma levels of P4 at the onset of oestrus were higher than normal, but it was concluded that the plasma levels of PGF2 alpha metabolite and P4 in RBH at luteal regression and early pregnancy were normal.     

Embryo collection in prepubertal gilts and attempts to develop an improved embryo transfer technique

Prepubertal gilts were treated with 1,500 iu equine chorionic gonadotrophin, followed 72 hours later by 500 iu human chorionic gonadotrophin (hCG), and inseminated 36 and 48 hours later. Embryos were collected at slaughter 168 hours after the hCG treatment. Blastocysts classified as 'good' or 'fair' were transferred to synchronised recipients, either by conventional surgical means or by a 'semi-endoscopic' approach, and the recipients were slaughtered four weeks later. Of 238 donor gilts, 98.4 per cent had responded with a mean (se) 23.5 (1.0) ovulations and 19.1 (1.0) ova or embryos, of which 47 per cent were considered morphologically intact and transferable. The large proportion of non-transferable embryos was not associated with the age or weight of the gilts, the season or with their housing conditions. Conventional surgical transfer of 15 to 20 (mean 17.4) blastocysts to synchronised recipients yielded 88 percent (14 of 16) pregnancies with between seven and 14 (mean 8.2) viable fetuses, and an embryo survival rate of 47 per cent in the pregnant recipients and 41 per cent in all the recipients. The corresponding data for the semi-endoscopic transfers were 16 to 20 (mean 17.7) blastocysts transferred, 47 per cent (eight of 17) pregnancies, four to 12 (mean 7.3) viable fetuses per pregnant recipient and an embryo survival rate of 41 per cent in the pregnant recipients and 19 per cent in all the recipients. Significantly fewer of these recipients became pregnant and a significantly smaller proportion of the embryos survived (P<0.05).     

Embryo transplantation in cattle. Non-surgical transfer of 6 1/2--7 1/2 day old embryos to lactating dairy cows under farm conditions.

Previous attempts to transfer embryos non-surgically to heifers have been rather discouraging. The present experiment describes a simple non-surgical technique where the 6½–7½ -day old embryo is placed in a 0.25 ml ministraw, fitted into a common insemination gun and transferred cervically to synchronized lactating dairy cows. Thirty-two viable embryos were transferred to the middle part of the uterine horn ipsilateral to the ovary containing the corpus luteum. It was easy to pass the cervix, and most embryos could be deposited in the horn within 1 or 2 min. Eighteen animals were diagnosed pregnant (56% ). It is conceivable that the easy atraumatic transfers and good management (feeding, oestrus control) of the recipients contributed to the high pregnancy rate.     

Re-insertion of a progesterone-containing intravaginal device to synchronise returns to oestrus in dairy heifers

Recommendations for oestrus synchronisation of dairy heifers using progesterone-containing intravaginal devices suggest re-insertion of used devices 16 days after first insemination for a period of 5 days to allow a second opportunity for artificial insemination. Controlled studies on the effectiveness of re-using intravaginal devices to synchronise returns to oestrus in non-pregnant dairy heifers are lacking. A clinical trial was conducted involving 750 Friesian heifers in 13 herds. After an initial synchronisation programme, the used intravaginal devices were re-inserted 14 or 16 days after first insemination into half of the heifers in each herd for a period of 5 days. After the first synchronisation programme, 47.5% of heifers remained non-pregnant. Re-insertion of used intravaginal devices for 5 days significantly increased the number of non-pregnant heifers detected in oestrus and inseminated by 48 hours after device removal compared to heifers in which devices were not re-inserted (45.2% v. 27.3%, p < 0.05, in herds where intravaginal devices were re-inserted on day 14; 48.8% v. 13.6%, p < 0.05, in herds where intravaginal devices were re-inserted on day 16). Re-insertion at 14 or 16 days after first insemination was equally effective in increasing visible returns to service. However, the number of non-pregnant heifers synchronised for a second round of artificial insemination was less than expected. Conception rate to the re-synchronised oestrus was unaffected by the treatment. It is concluded that the additional procedures of CIDR re-insertion, removal, tailpainting and insemination involved in there-synchrony programme, and the relatively low in-calf rate to the re-synchronised round of insemination, reduced the potential benefits of re-synchronisation.     

Effect of trypsin in semen on in vivo fertilization and early embryonic development in superovulated heifers

The effect of trypsin on the fertilizing capacity of bull semen was investigated as part of the evaluation of the addition of trypsin to semen as a method for destroying or inactivating infectious agents. Parts of the ejaculates from four bulls were treated with 0.3% trypsin solution. Both the treated and untreated aliquots of semen were frozen, thawed and used for the artificial insemination of superovulated heifers. Two hundred and thirty ova and embryos were collected from 22 heifers on day 7 after oestrus (insemination). One hundred and ten out of 164 (67%) embryos and ova from 15 heifers inseminated with trypsin-treated semen were classified as of transferable quality compared to 46 out of 66 (70%) in the control group of 7 heifers (p>0.05). There was no difference in the proportion of fertilized ova or degenerated embryos resulting from the control or trypsin-treated samples of frozen-thawed semen, which is consistent with results obtained previously using fresh semen.     

胚胎质量和发育阶段对奶牛胚胎移植妊娠率的影响

[目的]为了评估胚胎质量和发育阶段对奶牛胚胎移植妊娠率的影响。[方法]使用63头青年奶牛作为供体进行超数排卵,评估回收胚胎质量和发育阶段。选择334头青年奶牛作为受体鲜胚移植不同质量和发育阶段胚胎。对胚胎质量分布、发育阶段分布、不同质量胚胎和不同发育阶段胚胎移植30 d妊娠率进行统计分析。[结果]可用胚胎中A级胚胎比例(60.78%)显著高于B级和C级胚胎比例(36.70%和2.52%)(P<0.05);致密桑椹胚比例(54.36%)显著高于早期囊胚,囊胚和扩张囊胚比例(18.35%,25.0%和2.29%)(P<0.05)。A级和B级胚胎移植30 d妊娠率(63.55%和64.35%)显著高于C级胚胎移植30 d妊娠率(44.44%)(P<0.05);致密桑椹胚、早期囊胚、囊胚和扩张囊胚移植30 d妊娠率差异不显著(P<0.05),早期囊胚、囊胚移植30 d妊娠率高于致密桑椹胚、扩张囊胚移植30 d妊娠率(P<0.05)。[结论]选择不同发育阶段的A级和B级胚胎能够获得较高胚胎移植妊娠率,增加早期囊胚和囊胚阶段胚胎移植数量能够提高胚胎移植妊娠率。     

A comparison of two techniques for the synchronisation of oestrus in dairy heifers.

Seventy-four heifers in four separate breeding groups were allocated into two treatment groups for oestrus synchronisation. Group 1 was given a combination of an initial injection of norgestomet and oestradiol valerate together with a norgestomet ear implant left in place for nine days. Group 2 was given two injections of the synthetic prostaglandin luprostiol 10 days apart. The animals in group 1 were artificially inseminated once 48 hours after the removal of the implant and those in group 2 were inseminated once 72 hours after the second injection of luprostiol. Subsequently any returns were rebred by either AI or natural service. There was no significant difference between the numbers of animals in the two treatment groups which were diagnosed pregnant 33 to 35 days after insemination, although the implant treatment gave a higher overall proportion of pregnancies (70 per cent, 51 per cent). It also gave significantly smaller numbers of 'open' days over the whole of the breeding period. A study of individual animals by progesterone assay and investigation of ovarian structures by real-time ultrasound showed that some of them had unusual progesterone profiles but nevertheless became pregnant. It would appear that a corpus luteum may be responsive to prostaglandins even though it is secreting only a low level of progesterone.     

Enhancing Embryo Yield in Superovulated Holstein Heifers by Immunization Against Inhibin

Eight heifers, aged 16–17 months and showing normal oestrous cycles, were immunized against a recombinant porcine inhibin α subunit immunogen, together with another 10 heifers of the same age as controls and treated with placebo immunogen. Primary (1 mg immunogen) and two booster (0.5 mg immunogen each) immunizations were administered at 28‐day intervals. Ten days after the second booster immunization, both groups of heifers underwent a superovulation treatment. Each animal was given an intravaginal progesterone releasing sponge, which was withdrawn 7 days following an i.m. injection of 0.5 mg cloprostenol. Heifers were treated with FSH for 4 days and artificially inseminated after oestrus occurred. The embryos were flushed and evaluated 7 days after insemination. Immunization significantly (p < 0.01) increased blood antibody titres against recombinant porcine inhibin α subunit, from pre‐immunizaion and control values of approximately 0.06 of ELISA 450 nm reading to 0.6 to 0.7 after two or three immunizations. The immunized heifers produced on average 15.8 ± 2.8 embryos, significantly (p < 0.05) higher than the yield of 8.3 ± 1.5 in the controls. The number of transferable embryos were non‐significantly higher in immunized than in control heifers (9.6 ± 3.1 vs 5.8 ± 1.6, p > 0.05). The peak plasma oestradiol concentrations were significantly higher in immunized than in control heifers, both immediately after FSH treatment and 20 days thereafter. Plasma P4 concentrations after superovulation were in the range of 20 ng / ml in the immunized heifers, significantly (p < 0.05) higher than the values approximately 15 ng / ml in control heifers. These results indicated that prior immunization against inhibin α subunit stimulated production of antibodies against inhibin, which enhanced follicular developmental response to superovulation and lead to higher yield of total and transferable embryos. Therefore immunization combined with the conventional superovulatory gonadotrophin treatment, can be a simple and efficient method to produce low cost bovine embryos.     

Surgical and non-surgical transfer of bovine embryos

Methods of transferring one bovine embryo to the ipsilateral uterine horn have been compared. In heifers mid-ventral surgical laparotomy under general anaesthesia (n=22) was compared with flank surgery with paravertebral anaesthesia (n=21). Pregnancy rate was higher with a midventral approach (P less than 0.05) (77.3 per cent of 42.9 per cent respectively). In heifers mid-ventral surgery (n=36) was compared with two methods of non-surgical transfer either using the Cassou insemination gun (n=36) or a modification of it (n=39) with embryos collected on days 7, 8 or 10 after oestrus. There were no differences in pregnancy rate between methods or different ages of embryos (methods: 55.6, 55.6, 43.6 per cent respectively; Day: 48.8, 55.8, 48.1 per cent respectively) but the condition score of recipients affected success (P less than 0.05). Of 10 cows which each received an embryo using the modified Cassou gun eight became pregnant.