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.     

Technical note: a 2-stage cecal cannulation technique in standing horses

Cecal cannulation is necessary for sampling of intestinal contents for a variety of nutritional or digestive physiology studies. This report describes a 2-stage technique for permanent cecal cannulation in standing horses. For the first procedure, a right flank laparotomy is performed and a small pouch of the cecal base exteriorized and sutured to the body wall. The second procedure is performed approximately 1 wk later. During the second procedure, the exposed cecal pouch is removed and the cannula inserted. Ten horses were cannulated using this technique. After the first procedure, 1 horse developed a cecal impaction unresponsive to medical therapy and ruptured its cecum, whereas 2 other horses developed mild transient colic that responded to medical management. Insertion of the cecal cannula after creation of the stoma in the second procedure resulted in transient colic in 4 of 9 horses, but they responded to analgesic therapy in less than 24 h in all instances. The time to complete healing of the cannula site was approximately 30 d. The technique described in this report decreases the risk of peritonitis due to intestinal leakage and is technically easier to perform than previously described techniques.     

Technical note: a technique for multiple liver biopsies in neonatal calves

Our objective was to develop a rapid and safe liver biopsy technique that could be repeated on multiple occasions in individual neonatal calves. A pilot study was performed to verify the efficacy of sedation and restraint procedures and to evaluate different biopsy instruments. Following the pilot experiment, a biopsy trocar was fabricated and an experiment was conducted using this procedure. Liver biopsies were performed in neonatal calves on d 4, 9, 15, 21, and 28 of life to evaluate the effect of vitamin A intake on liver vitamin A concentrations. On these days, a single injection of ceftiofur sodium was administered i.m. 1 to 2 h prior to the procedure. Calves were lightly sedated with xylazine and placed on a surgical table in left-lateral recumbency. The right caudo-thoracic area was clipped and scrubbed with an iodophor agent. Following administration of a local anesthetic (lidocaine), a small incision was made in the skin between the 12th and 13th ribs approximately 15 cm from the dorsal midline. The biopsy trocar was inserted through the body wall and peritoneum and introduced into the liver parenchyma, and a liver sample was collected. Following the biopsy, the cutaneous incision was sutured and an antiseptic agent was applied to prevent infection. An i.m. injection of an analgesic was administered 1 h following the procedure to alleviate postsurgical discomfort. Most calves were able to stand within 2 h after the biopsy. The entire procedure, which could be performed by a single individual, usually required about 20 min from initial sedation until skin closure. Although liver samples of up to 500 mg were obtained, most samples weighed 75 to 150 mg (wet weight). A total of 156 liver biopsies were performed on 33 calves. Complications due to the biopsy procedure were observed in only two calves. Therefore, this procedure can be useful for studies designed to monitor changes in liver composition or enzyme activities over time.     

Technical note: a technique for ear vein catheterization in group-housed sows

No methods have been published for repeated blood sampling via an ear vein in group-housed sows. Therefore, the objective of this study was to develop a minimally invasive technique for the insertion of an ear vein catheter for repeated blood sampling in group-housed peripartum sows while minimizing any impact on production performance. Thirty-three multiparous pregnant sows were used including 18 catheterized sows and 15 control sows. In a group-farrowing barn, sows (8/room) shared a communal area and farrowed in individual, free-access pens. Treatment sows were anesthetized, and 1 ear was prepared aseptically 2 to 4 d before their expected farrowing date. A sterile needle was inserted into the largest and straightest portion of the vein, and the catheter, which was medical-grade microbore tubing, was inserted through the needle at least 24 cm. The needle was withdrawn, and the catheter was fixed into position and sutured to the ear. A blunt-end probe point cannula was glued onto the distal end of the catheter, and an adaptor injection cap with male Luer lock was placed on the end. The catheter was coiled and placed in a protective purse, which was cemented directly to the skin on the back of the shoulders. The catheter was flushed with heparinized saline to ensure patency. Once sows were able to stand, an elastic bandage was wrapped around the neck and upper body of the sow to hold the protective purse and exposed catheter in position. Blood samples were collected every 24 h, and catheters were flushed with heparinized saline after each collection. Fourteen of the 18 insertions were successful, and 11 of those remained functional for 4 d or more. Differences were not observed in reproductive performance between catheterized and noncatheterized sows.     

Technical note: Vitrification of goat embryos by the open pulled-straw method

The suitability of the open pulled-straw (OPS) method for vitrifying bovine embryos was tested for goat embryos. Of 14 does receiving OPS-vitrified embryos, all became pregnant and 13 (93%) kidded. The corresponding values for an established conventional freezing program were 58% pregnant and 50% (6/12) kidding. Overall embryo survival amounted to 64% (18/28) for OPS-vitrified and 42% (10/24) for conventionally frozen embryos. All differences were statistically significant. It is concluded that OPS vitrification is a suitable method for cryopreserving caprine d-7 blastocysts.     

Technical note: determination of alleles of the ovine PRNP gene using PCR-single-strand conformational polymorphism analysis

Susceptibility to scrapie in sheep is linked to variation at codons 136, 154, and 171 in the host prion protein gene (PRNP). A number of techniques are available for detecting these polymorphisms, but none allow for a rapid and accurate determination of genotype. Here we describe PCR coupled with single-strand conformational polymorphism (SSCP) analysis, which allows for the accurate identification of ovine PRNP alleles. A gene region including codons 136 to 171 was amplified by PCR, and the amplimers were then denatured and subjected to electrophoresis in a nondenaturing polyacrylamide gel. Nine unique SSCP patterns, representing nine different alleles of the ovine PRNP gene, could be resolved. A new polymorphism (I/T) at codon 142 also was detected. The profiles produced by SSCP allowed for the accurate differentiation of PRNP alleles and could be employed to genotype PRNP in sheep.     

A simplified biopsy method for precompacted mouse embryos: a technical report

This article presents a new, simple and rapid embryo biopsy method. The blastomere for genetic analysis can be separated from a precompacted mouse embryo after a partial zona digestion with the use of a holding pipette. For the micromanipulation only two microcapillaries and micromanipulators are needed. The development of the biopsied embryos was studied during in vitro culture and in utero following embryo transfer. There was no significant difference between the treated and the control groups in the ratio of embryos that developed to the blastocyst stage, although the biopsied embryos were delayed in their development because they contained significantly fewer cells compared to the control ones at the same stage. Although there was no difference in the ratio of implantation, the development of the biopsied embryos in utero was also delayed 12-24 hours on the 9th day of pregnancy. No difference in development was visible from the 13th day of pregnancy. Statistically, no differences were found in the developmental ratio (number of developed fetuses/transferred embryos) of the control and treated embryos during gastrulation (9th day of pregnancy), at the beginning of organogenesis (13th day of pregnancy) and before birth (19th day of pregnancy). The embryo biopsy method presented here can be a new and useful tool for preimplantation genetic diagnosis.     

Technical note: a double L intestinal cannula for cattle

A double L-shaped intestinal cannula was developed in an attempt to overcome problems observed previously with simple T-type cannulas. The cannula was constructed from cyclopolyvinyl chloride water pipe fittings. Construction materials were fairly rigid, but by connecting the split cannula pieces with elastic castration bands the cannula had some flexibility. Placing a short cone over the exposed cannula barrel reduced mechanical damage to the intestine. The double L cannula required a much smaller incision in the intestine during surgical insertion than a T-type cannula; it also simplified replacement. Construction is described; use and performance of the cannula has been satisfactory.     

Technical note: development of a duodenal cannula for sheep.

A single T-shaped duodenal cannula of silicone rubber with a gutter-type small inner flange was developed for sheep. The barrel of the cannula was 24 mm long with an internal diameter of 12 mm. A polyester surgical mesh (100 mm x 100 mm) was connected to the barrel of the cannula as an anchor. Fibrous tissues grew on the polyester mesh anchor and adhered firmly to the serosa of the intestine, thus leaving no gap and, hence, preventing any leakage of intestinal contents from the side of the fistula. The small (24 mm in diameter) and thin (3 mm in thickness) outer flange of the cannula became buried in the wool and prevented any mechanical disturbance of the cannula by the activity of the animal. The elasticity of the silicone rubber prevented distortion of the duodenum around the barrel of the cannula. No erosion of the tissue between the inner flange of the cannula and the mesh was seen in postmortem observations.     

Sexing of pre-implantation ovine embryos through polymerase chain reaction-based amplification of GAPDH,SRY and AMEL genes

The ability to identify the sex of embryo and control of sex ratio has a great commercial importance to livestock industry. Prediction of embryonic sex could be useful in the management decisions of sex selection in breeding programs. Several methods have been attempted to determine the sex but the polymerase chain reaction (PCR)-based sexing method is generally favoured, as it is cost effective, simple and reliable. The aim of the present study was to identify sex of sheep embryos produced in vitro through amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), sex-determining region Y (SRY) and amelogenin genes present in genomic DNA (gDNA) of embryos through PCR. To avoid false interpretation of the result by no amplification of SRY in female embryos, a duplex PCR was approached to amplify combinedly SRY and GAPDH genes. Sex-specific blood was used in PCR as positive control. In vitro sheep embryos were produced as per standardized protocol of laboratory. Sexing of sex-specific blood and in vitro produced embryos were approached though PCR to amplify the respective genes using gDNA present in the sample without its traditional isolation. The accuracy of sex prediction for embryos was 100% by this procedure.     

Technical note: A novel technique to assess internal body fat of cattle by using real-time ultrasound

The objectives of this study were to describe a system to assess KPH fat by using real-time ultrasound (RTU) and to develop equations to predict total physical separable internal fat (IFAT) based on ultrasound measurements. Data for this study were obtained from 24 Angus steers fed either hay- or corn-based diets during the backgrounding phase. Steers were serially slaughtered in 3 groups: at weaning (baseline), then at 4 and 8 mo after weaning. A fourth group was composed of 4 steers from the hay-fed group that were slaughtered at approximately 10 mo after weaning. The RTU measurements were collected every 2 mo, with a preslaughter scan approximately 7 d before the slaughter time. The RTU measurements consisted of 12th- to 13th-rib backfat thickness, 12th to 13th ribeye area, percentage of intramuscular fat, and kidney fat depth, which was measured in a cross-sectional image collected between the first lumbar vertebra and the 13th rib. For kidney fat, the ultrasound probe was placed on the flank region approximately 15 cm from the midline of the animal. Images were stored in the ultrasound console, and measurements were taken between the ventral part of the iliocostalis muscle and the end of the KPH fat at the chute side. The relationship between carcass and ultrasound measurements in the depths of kidney fat (cKFd and uKFd, respectively) had an r(2) of 0.93, with a root mean square error (RMSE) of 1.14 cm. An allometric regression between carcass KPH weight (cKPHwt) and cKFd was identified, and the untransformed regression had an r(2) of 0.96. The linear regression between total IFAT and cKPHwt had an r(2) of 0.97, with an RMSE of 2.67 kg. Therefore, a system was developed to predict IFAT from uKFd measurements by combining these equations. Additionally, a single linear regression between IFAT and uKFd measurements was developed (r(2) = 0.89, RMSE = 5.32 kg). Even though the system of equations had a lower RMSE of prediction and greater r(2) compared with the single linear regression (4.80 vs. 5.10 kg and 0.91 vs. 0.89, respectively), there was no difference between these methods in predicting IFAT (P = 0.4936) by using a pairwise mean square error of prediction analysis. Our results indicated that uKFd measurements can accurately and precisely predict the cKFd of steers consuming either high concentrate or forage rations. The results also showed that cKFd is highly correlated with cKPHwt, which can be used to estimate total IFAT. More research is needed to further evaluate this technique with different feeding strategies, breeds, and sexes.     

Proteomic profile of pre-implantational ovine embryos produced in vivo

The present study was conducted to decipher the proteome of in vivo-produced pre-implantation ovine embryos. Ten locally adapted Morana Nova ewes received hormonal treatment and were inseminated 12 hr after ovulation. Six days later, 54 embryos (morula and blastocyst developmental state) were recovered from eight ewes and pooled to obtain sufficient protein for proteomic analysis. Extracted embryo proteins were analysed by LC-MS/MS, followed by identification based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, PepExplorer). Identified proteins were analysed for gene ontology terms, protein clusters and interactions. Genes associated with the ovine embryo proteome were screened for miRNA targets using data sets of TargetScan ( http://www.targetscan.org ) and mIRBase ( http://www.mirbase.org ) servers. There were 667 proteins identified in the ovine embryos. Biological processes of such proteins were mainly related to cellular process and regulation, and molecular functions, to binding and catalytic activity. Analysis of the embryo proteins revealed 49 enriched functional clusters, linked to energy metabolism (TCA cycle, pyruvate and glycolysis metabolism), zona pellucida (ZP), MAPK signalling pathway, tight junction, binding of sperm to ZP, translation, proteasome, cell cycle and calcium/phospholipid binding. Sixteen miRNAs were related to 25 pre-implantation ovine embryo genes, all conserved in human, bovine and ovine species. The interaction network generated by miRNet showed four key miRNAs (hsa-mir-106b-5p; hsa-mir-30-5p; hsa-mir-103a-5p and hsa-mir-106a-5p) with potential interactions with embryo-expressed genes. Functional analysis of the network indicated that miRNAs modulate genes related to cell cycle, regulation of stem cell and embryonic cell differentiation, among others. Retrieved miRNAs also modulate the expression of genes involved in cell signalling pathways, such as MAPK, Wnt, TGF-beta, p53 and Toll-like receptor. The current study describes the first major proteomic profile of 6-day-old ovine embryos produced in vivo, setting a comprehensive foundation for our understanding of embryo physiology in the ovine species.     

A simplified one-step nuclear transfer procedure alters the gene expression patterns and developmental potential of cloned porcine embryos

Various somatic cell nuclear transfer (SCNT) techniques for mammalian species have been developed to adjust species-specific procedures to oocyte-associated differences among species. Species-specific SCNT protocols may result in different expression levels of developmentally important genes that may affect embryonic development and pregnancy. In the present study, porcine oocytes were treated with demecolcine that facilitated enucleation with protruding genetic material. Enucleation and donor cell injection were performed either simultaneously with a single pipette (simplified one-step SCNT; SONT) or separately with different pipettes (conventional two-step SCNT; CTNT) as the control procedure. After blastocysts from both groups were cultured in vitro, the expression levels of developmentally important genes (OCT4, NANOG, EOMES, CDX2, GLUT-1, PolyA, and HSP70) were analyzed by real-time quantitative polymerase chain reaction. Both the developmental rate according to blastocyst stage as well as the expression levels CDX2, EOMES, and HSP70 were elevated with SONT compared to CTNT. The genes with elevated expression are known to influence trophectoderm formation and heat stress-induced arrest. These results showed that our SONT technique improved the development of SCNT porcine embryos, and increased the expression of genes that are important for placental formation and stress-induced arrest.     

Diagnosis of ovine fascioliasis by a dot enzyme-linked immunosorbent assay: a rapid microdiagnostic technique

A microenzyme-linked immunosorbent assay (dot-ELISA) was modified for making an immunodiagnosis of Fasciola hepatica infections in sheep. Sheep were alloted as follows: group I-3 controls and 4 principals, each inoculated with 500 metacercariae; group II-3 controls and 7 principals, each inoculated with 250 metacercariae; and group III-3 controls and 7 principals, each inoculated with 500 metacercariae. Blood and fecal samples were collected from each animal every 2 weeks for 16 weeks. Presence (or absence) of flukes was confirmed by fecal examinations and examination of dissected livers at necropsy of the sheep. The dot-ELISA incubations were done at ambient room temperature. Nitrocellulose disks dotted with 1 microliter (50 ng of protein) of F hepatica excretory/secretory products were placed in 96-well tissue culture plates. After nonspecific binding sites on the disks were bound with bovine serum albumin-triethanolamine-buffered saline solution, dilutions (1:2) of positive- and negative-control serum samples or experimental serum samples were placed in appropriate wells for a 30-minute incubation. Wells were washed (3 times), and 50 microliters of horseradish peroxidase conjugated rabbit anti-sheep immunoglobulin G was added to each well for a 30-minute incubation and then aspirated. Substrate solution (4-chloro-1-naphthol, methanol, triethanolamine-buffered saline solution, and H2O2; 50 microliters) was added for a 30-minute incubation and then aspirated. Disks were air dried for visualization: solid purple dot = positive sample, or no dot = negative sample.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Technical note: a noninvasive procedure for measuring goat heart rates.

Heart rates were obtained simultaneously from FM radio transmitters and heart rate monitors externally mounted on unanesthetized and unrestrained mixed-breed goats. Data from transmitters were highly correlated (r = .92, P < .0001) with data from monitors and the percentage difference in heart rates between the two devices was less than that observed between animals. Analyses also revealed that radio transmitters provided a reliable, repeatable, and valid method for the noninvasive measurement of goat heart rates.     

Technical note: a device for obtaining time-integrated samples of ruminal fluid.

A device was adapted to allow for time-integrated sampling of fluid from the rumen via a cannula. The sampler consisted of a cup-shaped ceramic filter positioned in the ventral rumen of a cannulated cow and attached to a tube through which fluid entering the filter was removed continuously using a peristaltic pump. Rate of ruminal fluid removal using the device was monitored over two 36-h periods (at 6-h intervals) and was not affected (P > .05) by time, indicating that the system was not susceptible to clogging during this period. Two cows having ad libitum access to a totally mixed ration were used in a split-block design to evaluate the utility of the system for obtaining time-integrated samples of ruminal fluid. Ruminal fluid VFA concentration and pattern in samples collected in two replicated 8-h periods by the time-integrated sampler (at 1-h intervals) were compared with composite samples collected using a conventional suction-strainer device (at 30-min intervals). Each 8-h collection period started 2 h before or 6 h after feeding. Results indicated that total VFA concentration was not affected (P > .05) by the sampling method. Volatile fatty acid patterns were likewise unaffected (P > .05) except that acetate was 2.5% higher (P < .05) in samples collected 2 h before feeding and valerate was 5% higher (P < .05) in samples collected 6 h after feeding by the suction-strainer device. Although significant, these differences were not considered physiologically important. We concluded that use of the ceramic filter improved the sampling of ruminal fluid by simplifying the technique and allowing time-integrated samples to be obtained.     

Technical note: development of a transcervical oocyte recovery procedure for sheep.

An oocyte recovery procedure was developed and evaluated to determine whether a transcervical embryo recovery procedure is feasible with our method, which includes estradiol-17beta (E2) and oxytocin (OT) treatments, for dilating the cervix in ewes. On d 6 of an estrous cycle, oocytes were recovered either transcervically or with a laparotomy procedure. In the laparotomy group, ovulation rate was determined during the procedure and was used to calculate the percentage ofoocytes recovered. The laparotomy procedure was a standard uterine flush, and 12 mL of PBS was used to flush each uterine horn. In the transcervical group, the ovaries in each ewe were evaluated ultrasonically to determine ovulation rate. For transcervical recovery, 100 microg of E2 were injected i.v. on d 5 to increase cervical OT receptors, and 100 USP units of OT were injected i.v. 10 to 12 h later to dilate the cervix. Approximately 25 min after OT, ewes were placed in dorsal recumbency in a Commodore cradle, and a modified Foley catheter was passed through the cervix and into the uterus for injection (80 to 210 mL) and aspiration of PBS. The PBS was aspirated with a vacuum pump. The percentage of PBS recovered was greater (P<.01) at laparotomy than with the transcervical procedure (85.8 vs. 36.2%). Despite that difference, oocyte recovery did not differ significantly between the two groups (67% for laparotomy vs. 50% for transcervical; [oocytes recovered/number of corpora lutea] x 100), and there was no evidence that the transcervical procedure damaged the oocytes; the zona pellucida remained intact around all of the oocytes. In conclusion, a procedure that includes E2-OT-induced cervical dilation, passage of a modified Foley catheter into the uterus, and incremental infusion and aspiration of media through the catheter can be used to recover oocytes transcervically from ewes. This procedure may make transcervical embryo recovery feasible for sheep.     

Technical note: covariance adjustment in beef cattle research

In randomized experiments, analysis of covariance is used to increase precision of treatment comparisons. However, for factors that are observational (e.g., breed) or for covariates measured after treatments are applied, it may not be biologically meaningful to calculate treatment means adjusted to a common value of the covariate. For example, in beef cattle trials, it may not be meaningful to compare hot carcass weights of medium- and large-framed breeds adjusted to a common weaning weight because the breeds have naturally different mean weights at weaning. If done, this would typically result in an undesirable downward adjustment of mean carcass weight for the large-framed breed and upward adjustment of the mean carcass weight for the small-framed breed. However, it is desirable to evaluate the mean carcass weight for two diets, adjusted to a common weaning weight. Because of randomization, the expected weaning weights of animals on the two diets are equal and hence the only effect of covariance adjustment is to increase precision of the diet comparison. This paper presents the statistical methodology for estimating covariance adjusted means (termed partially adjusted means) when the levels of some of the factors are compared at a common value of the covariate but the levels of other factors are compared at differing values of the covariate. The methodology is extended to include several covariates, several factors, and arbitrary interactions among covariates, among factors, and between factors and covariates. These methods can be implemented using existing statistical software for linear models. Data are presented from an experiment in which hot carcass weight was recorded for beef cattle. Analyses of these data illustrate that adjusted means, partially adjusted means, and unadjusted means may differ substantially in magnitude, significance, and in the ranking of treatments.