Theriogenology

Several critical variables associated with successful cryopreservation of oyster embryos (Crassostrea gigas) were examined. These were 1) embryo developmental stage, 2) kind and concentration of cryoprotectant, 3) equilibration time, and 4) freezing rate. The percentage of survival was scored as the number of recovered embryos that swam actively 12 h after thawing and had developed into veliger stage. The oyster

Porcine handmade cloning (HMC), a simplified alternative of micromanipulation based traditional cloning (TC) has been developed in multiple phases during the past years, but the final evidence of its biological value, births of piglets was missing. Here we report the first births of ...

A tissue-culture system in which cells retain defined ultrastructural and functional characteristics was established to provide a basis for functional investigations of the epididymal duct in the cat. A widely used culture protocol for rat epididymal epithelium was used as a starting point and subsequently modified. The cellular population of the cat's epididymal epithelium was isolated by successive collagenase and trypsin digestion. A high yield of isolated cells obtained with good viability, were cultured in DMEM/F12 medium supplemented with foetal bovine serum, in absence or in presence of additional dihydrotestosterone (1 nM). The plated primary cultures reached confluence within 5-8 days, producing a monolayer of cohesive cells. Samples taken after 6 days in culture were processed for transmission and scanning electron microscopies. Immunocytochemical staining was used to estimate the purity of the epithelial cell population in the monolayers. The cell cultures displayed several functional traits of in vivo epithelia, including [ 35 S] hypotaurine and [ 35 S] taurine production. These results demonstrate that primary cultures of epididymal epithelial cells isolated from sexually mature cats maintain several differentiated characteristics of the intact organ and therefore provide a valuable system for the study of epididymal epithelial cell functions, metabolic activities and their regulation in cats. #

The dynamics of sperm DNA fragmentation (sDF) and sperm viability were analyzed in frozen-thawed sperm samples of Equus asinus (Zamorano-Leonés), a breed at risk of extinction. Sperm DNA fragmentation was assessed using an adaptation of the sperm chromatin dispersion test developed for stallions in five different frozen samples. Sperm were thawed and incubated at different temperatures (37 8C, 25 8C, and 4 8C) and sDF was assessed at different times and compared. The mean sDF after thawing at the beginning of the experiment was 18.20 AE 14.77% and did not differ significantly from the results of a neutral comet assay (22.0 AE 19.34%). The tendency in the sDF of all donkeys indicated that sperm DNA is more sensitive to breakage when incubated at 37 8C than when incubated at 25 8C or 4 8C. Interestingly, the tendency was not the same when different animals were compared, and differences in sDF dynamics were established among individuals. sDF correlated negatively with sperm viability in some individuals but not in others. From a conservation perspective, sDF analysis may offer a new way to assess sperm quality in endangered breeds in order to identify and select the best semen samples for artificial reproduction purposes. In particular, we recommend for artificial insemination the use of semen samples with a slow increase in sDF with time after thawing. #

Boar contact can induce ovarian activity, advance estrus and stimulate estrous behavior in sows. High amounts of boar contact can, however, suppress estrous behaviour. The present study with primiparous sows was designed to compare sows that had contact with a teaser boar during detection of estrus, with sows that had no boar contact at all. Number of sows detected in estrus within 9 d after weaning, onset and duration of e&us, follicular dynamics and timing of ovulation were studied. Boar contact increased the number of sows that ovulated and showed estrus from 14 of 47 to 24 of 47 (P < 0.05). Average timing of ovulation was later for sows with boar contact (165 h vs 150 h after weaning). Duration of estrus, detected without a boar, was similar in the two groups. For the sows with boar contact, duration of estrus detected with a boar was longer than estrus detected without a boar (56 vs 38 h; P < .Ol). Follicular dynamics were not affected by boar contact, boar contact only increased the munber of sows with ovulation. Ovulatory sows showed a larger increase in follicular diameter (P < 0.01) from weaning to Day 4 after weaning (from 2.3 to 5.4 mm) than anovulatory sows (from 2.5 to 4 mm). Anovulatory sows did not show follicular growth after Day 4. It is concluded that boar contact can increase the number of sows that ovulate and show estrus after weaning. Estrous behavior does not seem to be suppressed by contact with a teaser boar, compared to sows without boar contact. Q2oaobyEls&vlersclenceInc

The trend of increasing cow numbers per husbandry person makes it difficult to achieve good reproductive management by traditional means. A system to predict the reproductive status of cows would be of great value. There exist time-series models for detecting oestrus from milk traits other than progesterone and decision strategies for interpreting progesterone measures have been suggested but a system combining these aspects to predict not just oestrus but reproductive status throughout the reproductive cycle is lacking. The objective of this study was to develop such a model on the basis of automated milk progesterone sampling.

Progesterone is metabolized to pregnanediones and hydroxylated pregnanes prior to its fecal excretion. Therefore, use of progesterone antibodies underestimates the actual amount of fecal metabolites. To improve the methodology of noninvasive fecal progesterone metabolite analysis, enzymeimmunoassays (EIA) using group-specific antibodies against 5-reduced 20-oxo-pregnane-C3-conjugates were developed. Fecal and milk samples were collected at 1- to 2-d intervals during the morning and evening milking throughout 1 estrous cycle in dairy cows (n = 12). Six immunoreactive metabolites were detected in the feces with high performance liquid chromatography (HPLC), eluting as 5α- and 5β-reduced pregnanes containing a 20-oxo-group (20-oxo-pregnanes). Fecal samples of 3 cows were analyzed by 3 EIAs using antibodies against 4-pregnene-6α-ol-3,20-dione 6HS:BSA (6HS-progesterone), 5α-pregnane-3β-ol-20-one 3HS:BSA and 5β-pregnane-3β-ol-20-one 3HS:BSA, respectively. The follicular and luteal phases were identifiable with each EIA. Luteal phase values and the differences between mean follicular (Days 0 to 2 and 19 to 21) and luteal phase (Days 10 to 16) values obtained with the 5-pregnane EIAs were 3- to 4-fold higher than with the 6HS-progesterone EIA. Since results with the former 2 EIAs were almost identical, the remaining samples were only analyzed by the EIA for 5β-pregnane-3α-ol-20-one. Fecal 20-oxo-pregnane concentrations were parallel to milk progesterone values, but had a lag time of about 0.5 d; the coefficient of correlation (P < 0.001) was 0.76 (y = 155.2 × + 37.2). Fecal 20-oxo-pregnane concentrations during the follicular and luteal phase were 39.5 ± 2.2 and 341 ± 15.2 ng/g feces, respectively. In conclusion, fecal 20-oxo-pregnanes are significantly correlated to milk progesterone concentrations. They consist of several metabolites and compared to a 6HS-progesterone antibody, their evaluation was improved using antibodies against 5-reduced pregnanes.

The purpose of this study was to investigate the association between seropositivity for the most frequent Leptospira serovars and reproductive losses in sows in Brazil. Serum samples from 351 sows from 18 herds (in the state of Rio de Janeiro, Brazil) with low reproductive efficiency were tested (microscopic agglutination) for antibodies against serovars of Leptospira. Antibodies were detected in serum samples of 66.1% of all sows, most frequently serovar icterohaemorrhagiae (43.1%), followed by pomona (18.1%) and tarassovi (9.9%). Seroreactivity to icterohaemorrhagiae and pomona were associated (P < 0.05) with impaired reproductive performance (and substantial economic loss). Seroreactivity for pomona was associated (P < 0.05) with stillborn piglets and mummified fetuses, whereas seroreactivity to icterohaemorrhagiae was associated (P < 0.05) with the number of piglets born dead. #

The objective was to characterize testicular development in Blackbelly sheep, focusing primarily on Sertoli cell number. Lambs (n = 43) were allotted into eight groups, and surgically castrated at 0, 3, 6, 9, 12, 15,18 or 21 weeks of age (n = 4-6 lambs per group). Testes were fixed and paraffin-embedded, cross-sections (5 mm) were stained and evaluated with quantitative morphometry techniques. Testis weight increased at a greater rate between 9 and 15 weeks of age, which was associated with remarkable changes in testicular histology, including increases in tubular tissue volume, and tubule diameter and length. Spermatogenesis started in a period between 9 and 12 weeks, lumen and elongated spermatids were observed for the first time at 12 weeks (78% of the tubules) and 15 weeks (37% of the tubules), respectively. Total number of Sertoli cells (mean AE S.E.M.) increased steadily from birth (531 AE 76 Â 10 6 ) to 15 weeks (12,008 AE 1722 Â 10 6 ), with no changes afterwards. Sertoli cell number per gram of testicular tissue decreased as lambs were older, with the most remarkable change occurring between Weeks 9 and 12. An early increase in serum LH was observed at 6 weeks of age, with testosterone (T) increasing at Weeks 12 and 21. In conclusion, Sertoli cells maintained the capacity of proliferating from birth to 15 weeks of age in Blackbelly sheep; furthermore, the period of accelerated testis growth was associated with increased serum T concentration and with important changes in testicular morphology, as a consequence of the beginning and establishment of spermatogenesis and Sertoli cell maturation. #

Hair sheep ewes (St. Croix White and Barbados Blackbelly) were used to evaluate 3 methods of estrus synchronization for use with transcervical artificial insemination (TAI). To synchronize estrus, ewes (n = 18) were treated with PGF2¢ (15 mg, im) 10 d apart, with controlled internal drug release (CIDR) devices containing 300 mg progesterone for 12 d (n = 18), or with intravaginal sponges containing 500 mg progesterone for 12 d (n = 18). On the day of the second PGF2a injection or at CIDR or sponge removal, sterile rams were placed with the ewes. jugular blood samples were collected from the ewes at 6-h intervals until the time of ovulation, and daily for 16 d after estrus (Day 0). Plasma was harvested and stored at-20°C until LH, and progesterone concentrations were determined by RIA. There was no difference (P > 0.10) in time to estrus among the CIDR-, PGF2c¢-or sponge-treated ewes. All of the ewes in the CIDR group and 94.4% of the sponge treated ewes exhibited estrus by 36 h after ram introduction, while only 72.2% of PGF2a-treated ewes showed signs of estrus by this time (P < 0.06). The time from ram introduction to ovulation was not different (P > 0.10) among the CIDR-, PGF2c¢-or sponge-treated ewes. The time to the preovulatory LH surge was similar (P > 0.10) among CIDR, PGF2c¢ and sponge treated ewes. Progesterone levels through Day 16 after the synchronized estrus were not different (P > 0.10) among treatment groups. Hair sheep ewes (n = 23) were synchronized using PGF2c¢ and bred by TAI using frozenthawed semen 48 h after the second injection. The conception rate to TAI was 2/23 (8.7%) and produced 3 ram lambs. In a subsequent trial, 17 ewes were synchronized with CIDR devices and bred by TAI using frozen-thawed semen 48 h after CIDR removal, resulting in a conception rate of 52.9 % (9/17). It is possible to synchronize estrus in hair sheep using either CIDRs, sponges or PGF2c¢. Even though there were no significant differences in the timing of ovulation Or the LH surge among the treatment groups, a higher conception rate was achieved in ewes synchronized with CIDR devices during the second trial. This may reflect an increase in the skill level of the TAI technidan.

The pattern of growth and regression of ovarian follicles was characterized during 7 complete estrous cycles in 5 water buffalo by daily ultrasonographic examinations of the ovaries. Follicles 14 mm were measured and their relative locations within the ovary were determined to follow the sequential development of each individual follicle. Results indicated the presence of either one (n = 3 estrous cycles) or two (n = 4 estrous cycles) waves of follicular growth per cycle. Each wave was characterized by the development of 1 large (dominant) follicle and a variable number of smaller (nondominant) follicles. In the single follicular wave pattern, the wave started on Day 1.3 + 0.7 (Mean + SEM; estrus = Day 0). In the 2-wave pattern, first and the second waves started on Days 1.8 2 0.6 and 7.8 k 2.0, respectively, and the dominant follicle in the second wave was the ovulatory follicle. The maximal size and growth rate of the dominant follicle in the first and second wave of the 2-wave cycles did not differ significantly. However, the growth rate of the ovulatory dominant follicle in the 2-wave cycles was greater (P < 0.05) than that in the single-wave cycles. On the day of estrus, the ovulatory follicle was always the largest follicle present on either ovary. In conclusion, this study shows that in buffalo the development of ovarian follicles > 4 mm occurs in 1 or 2 waves per estrous cycle.

The objective of this study was to describe the sexual characteristics of domestic queens kept under natural equatorial photoperiod conditions without mating. Estrous signs were detected in 25 pubertal queens by manual stimulation and by exposure to a tomcat twice daily for 6 months (January to June). The signs observed were tail deflection, spinal flexion, rubbing or rolling, vaginal discharge, vocalization, treading of the hind legs, body or tail tremor and rigidity, blow or scratches, and discomfort on manipulation. The queen was considered in estrous when neck grip, tail deflection and attempted penile intromission by the male were allowed after mounting. From 187 cycles, there were (mean AE S.E.M.) 7.5 AE 0.7 cycles detected per queen; the duration of the cycle, estrus and non-acceptance were 18.1 AE 0.9, 7.9 AE 0.5, and 10.3 AE 0.9 d, respectively. Queens always maintained some signs of sexual behaviour; they remained ambivalent for no more than 24 h at a time. It was noted that 85.3% of the observations of body or tail tremor and rigidity were made during estrus; therefore, these signs were considered characteristic of sexual receptivity. There was no evidence of prolonged anestrus or of a circannual pattern to estrus cyclicity. #

The objective was to compare the use of powdered coconut water (ACP-109c; ACP Biotecnologia, Fortaleza, CE, Brazil) and Tris extenders for recovery and cryopreservation of epididymal sperm from agouti. The caudae epididymus and proximal ductus deferens from 10 sexually mature agoutis were subjected to retrograde washing using ACP-109c (ACP Biotecnologia) or Tris. Epididymal sperm were evaluated for motility, vigor, sperm viability, membrane integrity, and morphology. Samples were centrifuged, and extended in the same diluents plus egg yolk (20%) and glycerol (6%), frozen in liquid nitrogen, and subsequently thawed at 37°C for 1 min, followed by re-evaluation of sperm characteristics. The two extenders were similarly efficient for epididymal recovery, with regard to the number and quality of sperm recovered. However, for both extenders, sperm quality decreased (P Ͻ 0.05) after centrifugation and dilution. After sperm cryopreservation and thawing, there were (mean Ϯ SEM) 26.5 Ϯ 2.6% motile sperm with 2.6 Ϯ 0.2 vigor in the ACP-109c (ACP Biotecnologia) group, which was significantly better than 9.7 Ϯ 2.6% motile sperm with 1.2 Ϯ 0.3 vigor in Tris. In conclusion, agouti epididymal sperm were successfully recovered using either ACP-109c (ACP Biotecnologia) or Tris extenders; however, ACP-109c (ACP Biotecnologia) was a significantly better extender for processing and cryopreserving these sperm.

The primary objective of this paper is to review our current understanding of phenotypic variation in reproductive traits of AI boars. The proportion of boars that cannot be trained for collection in commercial studs is low and differences among genetic lines are small. In contrast, there is a considerable variation in sperm production and significant differences are present among genotypes. The general pattern is for sperm numbers to increase rapidly between 9 and 13 months of age and then gradually reach a plateau. This initial period of enhanced production occurs over a longer period in some genetic lines, resulting in differences of 30 Â 10 9 sperm cells or more per ejaculate. There also are genetic lines of boars that seem to have a high ''heat tolerance''. Decreases in sperm production during periods of high environmental temperatures average 5-7% in these lines, compared with 15-20% in others. Finally, there are boars currently being used in the industry that are capable of producing exceptional fertility results with low numbers of sperm. Unfortunately, several breeding practices common to swine AI make their routine identification difficult. Based on the phenotypic variation observed in modern terminal sire lines of AI boars, current prospects for influencing sperm production, boar fertility, and mounting behaviours through genetic selection are viewed as being good, moderate to low, and poor, respectively.

Multi-proxy approach was used to reconstruct the environmental conditions of remote lakes in the High Tatra Mountains (Slovakia) over the past few centuries (approximately 500-1000 years). Short sediment cores (*30 cm) taken from three morphologically similar glacial lakes distributed along altitudinal gradient (subalpine to alpine conditions) were analysed for organic matter content (LOI), diatoms and chironomids. Both descriptive and correlative approaches were used for analysing stratigraphical data. Predictive canonical correspondence analysis and co-correspondence analysis were applied to directly relate physical and biological proxies to each other. The relationship between LOI and biotic proxies was inconsistent across groups and lakes. Concordant patterns in diatom and chironomid composition were found in two non-acidified lakes. Common trends in those assemblages indicated major past environmental events such as the Little Ice Age, air pollution and lake acidification. In contrast, no relationship between the composition of diatom and chironomid assemblages was found in the formerly acidified lake, suggesting different responses of assemblages to acidification. While chironomids showed shifts that are attributable to recovery, diatoms assemblage remained relatively stable throughout the uppermost layers of the sediment record. On the other hand, climatic-driven changes in assemblages detected in the deeper layers were more pronounced in diatoms than in chironomids.

The isolation of pluripotent stem cells domestic animals will have a big influence on genetic modification of livestock. Pluripotent stem cell lines have been established for several domestic species. The in vivo pluripotency properties of stem cell lines has been demonstrated for bovine, sheep, and pig stem cell lines. Bovine phxipotent stem cell lines derived from embryos or primordial germ cells are an unliited source of pluripotent cells. Nuclear transfer of bovine pluripotent stem cells could play an important role in the production of offspring of domestic animals. However, placental development of fetuses derived from nuclear transfer of stem cells is incomplete, several approaches to overcome this problem described. Pluripotent stem cells will become valuable after demonstration of germ line transmission resulting in a full term live calf

Sex preselection of livestock offspring in cattle represents, nowadays, a big potential for genetic improvement and market demand satisfaction.Sperm sorting by flow cytometer provides a powerful tool for artificial insemination and production of predefined sexed embryos but, an accurate verification of the yield of sperm separation remains essential for a field application of this technique or for improvement and validation of other related semen sexing technologies.In this work a new method for the determination of the proportion of X- and Y-bearing spermatozoa in bovine semen sample was developed by real time PCR. Two sets of primers and internal TaqMan probes were designed on specific X- and Y-chromosome genes. To allow a direct quantification, a standard reference was established using two plasmid cDNA clones (ratio 1:1) for the specific gene targets. The method was validated by a series of accuracy, repeatability and reproducibility assays and by testing two sets of sorted and unsorted semen samples. A high degree of accuracy (98.9%), repeatability (CV = 2.58%) and reproducibility (CV = 2.57%) was shown.The results of X- and Y-sorted semen samples analysed by real time PCR and by flow cytometric reanalysis showed no significant difference (P > 0.05). The evaluation of X-chromosome bearing sperms content in unsorted samples showed an average of 51.11 ± 0.56% for ejaculates and 50.17 ± 0.58% for the commercial semen. This new method for quantification of the sexual chromosome content in spermatozoa demonstrated to be rapid and reliable, providing a valid support to the sperm sexing technologies.

In this study we compared the effects of preculture storage of ovaries, IVM medium, a reduced O 2 atmosphere and duration of culture on in vitro maturation (IVM) of domestic cat oocytes. One randomly selected ovary of each pair (69 pairs) was stored in PBS at 10 8C for 16±24 h before oocyte recovery. The second ovary from each pair was used as a nonstored control. In Experiment I, we investigated the effect of culture media (TCM 199 versus SOF) and a reduced O 2 atmosphere (a humidi®ed gas atmosphere of either 5% CO 2 in air or 5% CO 2 :5% O 2 :90% N 2 ) on IVM of both stored and nonstored oocytes. In the second experiment, we compared timing of nuclear maturation of both stored and nonstored oocytes cultured for 17±18, 20±21, 24±26, 28±30, 33±34 or 42±45 h before being evaluated for meiotic status. In both, Experiments I and II, the recovery rate, quality and competence for maturation of oocytes originating from stored ovaries did not differ (P > 0:05) compared with nonstored. In Experiment I, neither culture medium (37.5 versus 43.2% of Metaphase II, respectively in TCM 199 versus SOF) or gas atmosphere (40.0 versus 32.5% of Metaphase II, respectively in 5% CO 2 in air versus 5% CO 2 :5% O 2 :90% N 2 ) affected oocyte maturation. In Experiment II, the mean proportion of oocytes achieving Metaphase II within 17±18 h of culture was 36.1% and did not signi®cantly increase (P > 0:05) over time up to 28 h. The highest proportion of oocytes (67.3%) reached Metaphase II stage after 42±45 h of culture. Therefore, we conclude that two``waves'' of nuclear maturation of cat oocytes can be distinguished. The ®rst wave takes place within 26 h and it is likely that most oocytes of this wave mature by 17±18 h; the second wave occurs after 28±30 h of IVM. It can be assumed that this double wave may re¯ect the presence of two Theriogenology 59 (2003) 1567±1574 (L. Ka Îtska-Ksia Îz Çkiewicz). 0093-691X/02/$ ± see front matter # 2002 Published by Elsevier Science Inc. PII: S 0 0 9 3 -6 9 1 X ( 0 2 ) 0 1 2 0 0 -1 oocyte populations with two different degrees of``prematuration'' which require different lengths of IVM. #

The application of assisted reproductive technology (ART) to conservation biology has tremendous potential in the management and preservation of germ plasm from nondomestic ungulate species. Traditional approaches of superovulation and nonsurgical embryo recovery have been hampered in these species by inconsistent responses to commercially available gonadotropin preparations and by substantial interspecific variations in the details of reproductive regulatory processes. Although our general knowledge of reproductive physiology is improving, it appears that the production of embryos by IVF will more efficiently advance wildlife conservation efforts. This technology is already proving to be a powerful tool for rescuing gametes (sperm and oocytes) directly from the gonads of wildlife after death or gonadectomy. Other advanced procedures, like sperm microinjection and assisted hatching, may prove necessary for optimizing in vitro embryo production and in vivo developmental competence. Finally, there is a need to direct research attention to pathogen interactions with the zona pellucida (i.e. zona adherence and washing practices) to enhance the approved importation of valuable genetic material from free-ranging animals.

Data on breeding soundness and libido evaluations in Belgian Blue (BB) bulls are scarce. The present study compared results of breeding soundness and libido evaluations of young BB bulls to young Holstein Friesian (HF) bulls prior to acceptance into an AI program. Breed differences for breeding soundness exist between BB and HF bulls, as 93.7% of the young BB bulls failed the breeding soundness examination (BSE) compared to 59.3% of the HF bulls (P = 0.0005). Within the BB breed, differences were present between bulls of different ages, and bull selection for better fertility with increasing age apparently influenced the results. The number of reasons for which bulls failed the test differed between the age groups in the BB breed, whereas a tendency for more failure reasons in the BB breed was noticed in the breed comparison. The most important reasons for failure were sperm morphology and scrotal circumference (SC), but far more BB bulls failed for these traits compared to the HF breed (82.8% versus 56.0% and 43.8% versus 17.6% in the BB and the HF breed for sperm morphology (P = 0.0005) and SC (P < 0.0001), respectively). The high proportion of BB bulls with a substandard SC and poor sperm morphology might suggest an increased prevalence of testicular hypoplasia or degeneration within this breed. Concerning libido, the reaction time did not differ either between breeds or between age groups within the BB breed, whereas mounting enthusiasm, although not different between the two breeds, did decline with increasing age, probably due to the greater mating experience of the older bulls. All in all, libido did not seem to be different between the breeds.

The aim of the investigation was to determine the cause of infertility in 7 previously fertile bitches referred to the Small Animal Clinic University of Nairobi. Clinical, vaginal cytology and bacteriological evaluation of the bitches were performed during proestrus. Based on vaginal cytology, four of the bitches presented with prolonged proestral bleeding and were found to be in the fertile period of the oestrous cycle (80% "superficial" cells) although clinically presenting as being in proestrus based on the nature of vulval discharge. These bitches had been presented for breeding based on vulval discharge and were infertile due to improper timing of mating. Infertility in two other bitches was associated with Brucella canis primarily, and probably beta-hemolytic Streptococci super -infections. Infertility in the remaining bitch was due to the stud dog owner's assumption that mating had occurred after the stud dog and the aggressive bitch were put in the same kennel for the duration of the bitch's fertile period. The authors recognized that most breeders and some veterinarians did not understand the physiology of the canine oestrous cycle resulting in the misdiagnosis of causes of infertility in healthy bitches. This article highlights the challenges met when investigating infertility and the importance of vaginal cytology in breeding of bitches in Kenya.

Indications for estrus induction in the bitch include missed breeding opportunities or conception failure, the treatment of primary or secondary anestrus and synchronization of ovulation for embryo transfer programs. Reported methods for canine estrus induction include the use of synthetic estrogens (diethylstilbesterol), dopamine agonists (bromocryptine and cabergoline), GnRH agonists (lutrelin, buserelin, fertirelin, deslorelin, and leuprolide) and exogenous gonadotropins (luteinizing hormone, follicle stimulating hormone, human chorionic gonadotropin, pregnant mare serum gonadotropin, and human menopausal gonadotropin). These methods vary widely in efficacy of inducing estrus, as well as in the fertility of the induced estrus. The applicability of some of these methods for clinical practice is questionable. This review will summarize published reports on estrus induction and synchronization in bitches and summarize preliminary results using a long-acting injectable preparation of deslorelin.

Transcervical embryo collection is used routinely in the bovine species throughout the world to collect Day 6 to Day 9 embryos (early embryos) for genetic selection. For research purposes, however, the collection of embryos at later stages of pregnancy, i.e., Days 12 to 21 (late embryos), is needed. So far, for the recovery of late embryos, females are euthanized and embryo collection is performed after recovery of the genital tract. To reduce the number of animals used and still provide valuable material for embryo research, we have therefore developed a transcervical technique to collect late embryos. The objective of this study was to compare embryo recovery results at early and late stages within our laboratory. Altogether, 232 cows were used for this study. One hundred forty-five flushes were performed to collect embryos from Days 6 to 9, and 251 flushes were performed to collect embryos from Days 12 to 21. For the early embryos, a classical three-way collection equipment was used. To collect the late embryos, the same equipment was used, but the extensible flexible catheter that goes inside the external rigid catheter was removed, so that larger embryos could be collected through the remaining larger hole (two-way collection). All females were submitted to ovum pick up to remove the dominant follicle and were subsequently superovulated with FSH. Luteolysis was induced 48 hours before artificial insemination. Two artificial inseminations were performed with frozen semen, 48 and 56 hours after PGF2a injection. Before embryo collection, cows were treated with an epidural injection of a local anesthetic drug. The presence of CL was checked, and they were counted by rectal palpation. For all collections, the cervix was prepared with the initial introduction of a dilator. Then, the catheter was introduced in one horn, and the cuff was inflated as low as possible. For the collection of late embryos, the flushing solution (30 mL) was injected slowly twice to suspend the embryos before flushing the horn with 500 mL, and the same operation was performed on the second horn. There was no significant difference in the number of embryos collected per flush in the early-and latestage (758 embryos collected, 5.22 AE 6.02 per flush vs. 1238 embryos collected, 4.93 AE 5.07 per flush, respectively). The number of embryos collected per CL, however, was significantly lower in the early versus late group (0.39 AE 0.32% vs. 0.44 AE 0.34%, respectively). The late collection allowed the retrieval of full conceptuses (embryonic and extraembryonic tissues), even at very late stages such as Days 18 to 21. Careful collection is needed, however, so that conceptuses are not damaged or torn: the horn must be * Corresponding author. Tel.: þ33 1 34 65 24 19; fax: þ33 1 34 65 23 64. Theriogenology xxx (2015) 1-9 massaged gently and the flush should be ideally recovered in one single flow. This technique is a powerful tool to collect the late-stage embryos for research purposes. Because it is not traumatic, animals can be used again for the same procedure.

The effects of estradiol benzoate (EB) and estradiol cypionate (EC) on induction of ovulation after a synchronized LH surge and on fertility of Bos indicus females submitted to timed AI (TAI) were evaluated. In Experiment 1, ovariectomized Nelore heifers were used to evaluate the effect of EB (n ϭ 5) and EC (n ϭ 5) on the circulating LH profile. The LH surge timing (19.6 and 50.5 h; P ϭ 0.001), magnitude (20.5 and 9.4 ng/mL; P ϭ 0.005), duration (8.6 and 16.5 h; P ϭ 0.001), and area under the LH curve (158.6 and 339.4 ng/mL; P ϭ 0.01) differed between the EB and EC treatments, respectively. In Experiment 2 (follicular responses; n ϭ 60) and 3 (pregnancy per AI; P/AI; n ϭ 953) suckled Bos indicus beef cows submitted to an estradiol/progesterone-based synchronization protocol were assigned to receive one of two treatments to induce synchronized ovulation: 1 mg of EB im 24 h after progesterone (P4) device removal or 1 mg of EC im at P4 device removal. There was no difference (P Ͼ 0.05) between EB and EC treatments on follicular responses (maximum diameter of the ovulatory follicle, 13.1 vs. 13.9 mm; interval from progesterone device removal to ovulation, 70.2 vs. 68.5 h; and ovulation rate, 77.8 vs. 82.8%, respectively). In addition, P/AI was similar (P Ͻ 0.22) between the cows treated with EB (57.5%; 277/482) and EC (61.8%; 291/471). In conclusion, despite pharmacologic differences, both esters of estradiol administered either at P4 device removal (EC) or 24 h later (EB) were effective in inducing an LH surge which resulted in synchronized ovulations and similar P/AI in suckled Bos indicus beef cows submitted to TAI.

Multiple ovulation and embryo transfer (MOET) has the potential to increase the rate of genetic improvement in sheep. However, better realization of this potential requires maximum survival rates of transferred embryos of high genetic merit after transfer into recipient ewes. These studies were therefore conducted to investigate the effect of both embryonic and recipient ewe factors on the survival rate of transferred embryos. Survival rate was similar after transfer of morula or blastocyst stage embryos, and these were higher (P < 0:05) than for very early morulae and early morulae. Advanced embryos (Day 5 blastocyst) had an advantage (P < 0:05) in survival rate over retarded embryos (Day 6 morula). Grades 1 and 2 embryos survived signi®cantly (P < 0:05) better than Grades 3 or 4 embryos. There was no difference in embryo survival rate following transfer to recipients with different numbers of corpora lutea. In general, age or parity of recipient ewes did not affect embryo survival rate, although a higher (P < 0:05) embryo survival rate was observed for yearling recipients. Buserelin (GnRH agonist) treatment of recipient ewes 5 or 6 days after transfer of embryos (Day 12 of the cycle) did not improve embryo survival rate. These results con®rm that both embryonic and recipient factors can play an important role in the success of a MOET program in sheep. #

The Catalonian donkey breed is in danger of extinction, and much needs to be learned about the reproductive features of its females if breeding and conservation programmes are to be successful. This study reports the oestrous behaviour, oestrus cycle characteristics and dynamic ovarian events witnessed during 50 oestrous cycles (involving 106 ovulations) in 10 Catalonian jennies between March 2002 and January 2005. These jennies were teased, palpated transrectally and examined by ultrasound using a 5 MHz linear transducer-daily during oestrus and every other day during dioestrus. Predictors of ovulation were sought among the variables recorded.

The uterotonic characteristics and effectiveness of a single treatment with either oxytocin or carbetocin were quantified in early postpartum dairy cows after normal, uncomplicated calvings. Both the short-term (within 4 h), and the long-term effects (between 12 and 36 h) of the two treatments were compared. Between 14 and 16 h after parturition, 27 multiparous Holstein-Friesian dairy cows, without fetal membrane retention, were selected and divided into three groups. The first group (n = 9) was administered 50 IU oxytocin intramuscularly, the second group (n = 10) received 0.35 mg carbetocin, while animals of the third group (n = 8), serving as a control, were administered 5 mL saline solution. A transcervically introduced open tip catheter system was used for the non-invasive acquisition of the intrauterine pressure (IUP) recording. After digitalization, the signals were analyzed, using a specially adapted graphical software program. A significant short-term effect was found both ...