A simplified technique for the transfer of ovine embryos by laparoscopy

Embryos collected from ewes on days 5, 6 and 7 after oestrus were transferred to synchronised recipient ewes by a simple laparoscopic technique which resulted in a pregnancy rate of 75 per cent. The reduced surgical trauma and operative time have benefits in terms of animal welfare as well as for the economics of the embryo transfer industry.     

Laparoscopic artificial insemination in sheep

The goal of any AI program is to create improved offspring, and the achievement of this objective will depend on the breeding value of the ram and ewe selected. Laparoscopic AI is being utilized in the sheep industry to extend the use of superior rams, and it offers the producer the opportunity to maximize the reproductive potential of superior sheep. Rapid genetic trait infusion of known superior stud rams into the flock is the primary economic benefit of laparoscopic AI. The success of laparoscopic AI depends on events and factors that interrelate in a complex way. Once the selection and preparation of the ewe have been accomplished, one of the more important steps in the program is the successful synchronization of the ewe to deliver the necessary ova to the site of fertilization at a specific time. One of the best methods of synchronization for laparoscopic AI is the use of a progesterone product for a controlled time period and the administration of PMSG upon its removal. Detecting the onset of estrus is critical, and the addition of sterile (e.g., vasectomized) males is helpful, even essential, to accurately determine when each ewe begins her estrus. The ram effect has been shown to stimulate ovulation and estrus. Ewes must be inseminated within a narrow window of time after the synchronization product is removed. Ewes should be inseminated in the order in which they begin to exhibit signs of behavioral estrus, but age, stage of lactation, duration of behavioral estrus, and breed must be taken into account when this order is established. Fresh-extended semen works well throughout this preferred time frame established for laparoscopic AI, but frozen semen gives best results when used near the end. Advancement in manufacturing technology today removes equipment as a variable factor. It is important, therefore, that the inseminator develop a level of expertise in laparoscopy to ensure maximum fertilization rates. If available, fresh-extended semen is preferred over frozen semen, using at least the minimal number of spermatozoa necessary for fertilization. Evaluation of the post-thaw frozen or fresh semen is necessary to determine motility, morphology, and concentration, all of which help determine the volume of the insemination dose. The minimum necessary for laparoscopic AI in fine-wooled breeds is 20 X 10(6) normal motile spermatozoa; however, the more seasonal and less fertile American sheep need approximately 40 to 50 X 10(6) normal motile sperm to achieve acceptable fertility rates.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transfer of frozen-thawed embryos in sheep

Embryos collected from ewes six days after oestrus were frozen in straws using ethylene-glycol as a cryoprotectant. The efficiency of the simplified freezing and thawing procedure was assessed after transfer, which resulted in an overall survival rate of 58.3 per cent. Forty-two lambs were born from 72 frozen embryos which had been transferred without any attempt to evaluate them after the thawing and sucrose dilution process.     

Technical note: transfer of ovine embryos through a simplified mini-laparotomy technique

The aim of this experiment was to examine whether a simplified mini-laparotomy technique is suitable for embryo transfer in ewes. In vitro produced blastocysts were transferred to the uterine horns of synchronous recipient ewes. Each recipient received 1 embryo by conventional laparotomy (n = 36), laparoscopy (n = 21), or by simplified mini-laparotomy (n = 33). Pregnancy rates for these 3 transfer techniques were 38.9, 47.6, 45.5%, respectively (P = 0.58). Of these techniques, the simplified mini-laparotomy was preferred because of benefits it provided in terms of savings in time and expense as well as reductions in surgical trauma and elimination of exteriorization of most of the reproductive system along with only one suture. It is likely that this technique will play a substantial role in adoption of embryo transfer by the sheep industry.     

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.     

A technique for sampling total rumen contents in sheep

The paper describes the manufacture of an easily removable large rumen fistula closure for sheep and a restraining cradle which is used when sampling total rumen contents. Eight sheep have been fitted with the fistula and have been used regularly for nine months. The technique is rapid and simple and the closure is more reliable than other types which are often removed accidentally.     

A technique for the quantification of Duddingtonia flagrans chlamydospores in sheep faeces

Previous observations showed that Duddingtonia flagrans chlamydospores were visualized in McMaster chambers containing faeces of treated sheep. This trial explored the McMaster technique as a tool to quantify chlamydospores in sheep faeces. A range of individual chlamydospore doses (from 19.5 x 10(6) to 177.5 x 10(6)) were offered orally to nine lambs for 7 consecutive days. A faecal sample (5 g) was daily obtained from the rectum of each animal (from days 1 to 13) to perform the McMaster technique using a sugar flotation fluid with 1.27 g/mL density. Each chlamydospore counted in the McMaster chamber was considered as 50 chlamydospores per g of faeces (CPG). The results confirmed that the estimated CPG was associated with the daily dose offered to the animals (r(2)=0.90; P<0.001). Furthermore, the total chlamydospore dose received by each animal was strongly associated to the total quantity of CPG obtained from the bulk faeces (TCtot) (r(2)=0.96; P<0.0001). Quantification of CPG can be used as a helpful tool to determine the number of chlamydospores reaching the faeces in orally dosed animals. This could be used to evaluate the efficacy of D. flagrans for the control of gastrointestinal nematode larvae in sheep faeces.     

Effects of recipient oocyte source,number of transferred embryos and season on somatic cell nuclear transfer efficiency in sheep

To improve the efficiency of somatic cell nuclear transfer (SCNT) in sheep, we investigated the effects of recipient oocyte source, number of transferred embryos and season on the pregnancy and live lamb rates for sheep somatic cell nuclear transfer embryos. Follicle‐stimulating hormone (FSH)‐stimulated ovaries produced significantly more oocytes both in total and of suitable quality for maturation culture than those without FSH treatment (from slaughterhouse). However, their in vitro maturation rates were similar. Embryos were reconstructed using adult fibroblast cells into enucleated MII oocytes. The pregnancy and term rates were significantly higher in the FSH‐stimulated group than in the slaughterhouse one. Oocytes from FSH‐stimulated ovaries were enucleated as recipient cytoplasm for nuclear transfer in the following experiments. The transfer of 7–9 and 11–13 embryos produced significantly higher pregnancy rates than that of six embryos. However, the former groups exhibited similar live lamb rates. FSH‐stimulated ovaries produced significantly more oocytes in November and December (winter) than in May to July (summer), but the associated maturation rate did not increase. Pregnancy and term rates were significantly higher when transfer occurred in winter than in summer. In conclusion, FSH treatment produced significant benefit regarding the number and quality of collected oocytes and also for the pregnancy and live lamb rates for reconstructed embryos. However, the transfer of an appropriate number of embryos (7–13) and at an appropriate season (winter) increased pregnancy and term rates.     

A modified technique for flushing ova from the sheep uterus

Abstract

Extract

Sheep ova are normally obtained from the reproductive tract using the flushing techniques described by Hunter et al. (1955) Cumming, I. A. and McDonald, M. F. 1967. The production of ova by New Zealand Romney ewes following hormonal stimulation. N.Z. Jl agric. Res., 10: 226–236. [Taylor &; Francis Online], [Web of Science ®] , [Google Scholar]. Oviduct flushes, in the writers' experience, give a higher recovery of ova than uterine flushes, but they result in more adhesions of the tract, which can cause infertility. Consequently, the development of satisfactory uterine flushing techniques appears necessary before ova can be recovered repeatedly from superior ewes. This paper describes such a technique.     

Morphometric study of the testis in sheep embryos using unbiased design-based stereology

Sheep have been used as translational models of human postnatal testicular development. However, the morphometric features of the normal developing testis in sheep embryos have not been previously investigated using stereology. The objective of the present work was to establish normal quantitative parameters for fetal testicular tissue components in sheep, using unbiased design-based stereological methods. Twenty-four sheep embryos were divided into four gestational age groups (9–11, 12–14, 15–17 and 18–20 weeks of gestation) on the basis of the embryos’ crown-rump length. Isotropic, systematic uniform random sections of the left testes were obtained by employing the orientator method. Testicular total volume, the absolute and proportional volumes occupied by the seminiferous tubules and interstitial tissue, as well as the seminiferous tubule length, were estimated using the point-counting system and the unbiased counting frame principle. All the parameters, with the exception of the interstitial tissue's fractional volume, gradually increased along with gestational age, with the maximum increase especially seen in the late fetal stages. The proportional volume of the interstitial tissue, on the other hand, showed a decreasing trend along with increasing gestational age. The absolute volume of the testes, of the seminiferous tubules and of the interstitial tissue, and the length of the seminiferous tubules showed a significant (p< 0.05) positive linear correlation with gestational age. Several similarities were observed with human testicular embryogenesis. The stereological data emerging from the present study might prove useful as basic contribution to the fields of andrology and embryology and stimulate further research in these areas.     

New transtracheal bronchoalveolar lavage technique for the diagnosis of respiratory disease in sheep

A new transtracheal bronchoalveolar lavage technique for the diagnosis of respiratory disease in sheep under field conditions was tested in 76 sheep. The sheep were divided into three groups, normal sheep, sheep with clinical signs of respiratory disease and housed sheep, on the basis of their respiratory disease history and husbandry conditions. The detection of Mannheimia haemolytica and Mycoplasma ovipneumoniae or parainfluenza virus type 3 and bovine respiratory syncytial virus antigen in the lavage samples was closely correlated with clinical disease. The sheep with clinical respiratory disease had a higher mean percentage of neutrophils in the lavage fluid than the sheep in the other two groups.     

Pregnancy rate after fixed‐time transfer of cryopreserved embryos collected by non‐surgical route in Lacaune sheep

This study investigated the feasibility of applying fixed‐time (cryopreserved) embryo transfer in ewes. Embryos (n = 106) were non‐surgically recovered from superovulated donors (n = 39) on day 6–7 after oestrus. Straws containing one or two embryos (morulae and/or blastocysts) subjected to either slow freezing (SF, n = 62) or vitrification (VT, n = 44) were randomly used within fixed‐time embryo transfer on Day 8.5. Recipient ewes were nulliparous (n = 58) bearing corpora lutea after synchronous oestrous induction protocol. The pregnancy rate was higher (p = .03) in SF (39.4%) than VT (16.9%) and survival rate tended (p = .08) to be higher in SF than in VT (25.8% vs. 15.9%). Lambing rates were similar (p = .13) between SF (20.9%) and VT (15.9%). Embryos recovered by non‐surgical route after cervical dilation treatment and later cryopreserved by either slow freezing or vitrification produced reasonable pregnancy rates after FTET.