Showing papers in "Reproduction in Domestic Animals in 2002"

Influence of puerperal uterine infection on uterine involution and postpartum ovarian activity in dairy cows.

Abstract: This article presents the results of a clinical trial designed to study the effect of puerperal uterine infection on uterine involution and on ovarian activity in dairy cows, monitored twice weekly from parturition until the sixth week postpartum (wpp). Infection significantly retarded uterine involution assessed by the uterine body diameter and a score of intrauterine fluid volume (IUFV). By the sixth wpp, cows with normal puerperium (controls) and cows that showed mild puerperal endometritis had similar uterine body diameter and IUFV, indicating spontaneous recovery within the postpartum voluntary waiting period. However, in cows with severe puerperal endometritis, although uterine body diameter had regressed to pregravid size, IUFV remained significantly higher than in control and mild endometritis cows, indicating that chronic endometritis was established. The IUFV score was positively and significantly correlated with uterine swab bacterial growth density and allowed diagnosis of endometritis after the third wpp. Cows with mild or severe endometritis had a significantly higher prevalence and persistence of pathogenic bacteria (Escherichia coli, Actinomyces pyogenes, Gram-negative anaerobes - GNA) than controls. Actinomyces pyogenes was associated to GNA in 74% of isolations. Ovarian activity measured by ultrasound scanning of the ovaries and plasma progesterone (P4) concentrations was more abnormal (prolonged anoestrus, prolonged luteal phases and ovarian cysts) in cows with severe endometritis than in controls.

Formation of a Reservoir of Sperm in the Oviduct

Abstract: Contents A reservoir of sperm in the initial segment of the oviduct has been found in several species of domestic and laboratory mammals. Evidently, the reservoir serves to ensure successful fertilization by providing the appropriate number of sperm in the appropriate physiological state for fertilizing oocytes soon after they enter the oviduct. Recent evidence indicates that sperm are trapped in the reservoir by binding to specific carbohydrate moieties on the surface of the mucosal epithelium of the oviduct. A bovine seminal plasma protein has been identified that associates with sperm and confers on them the capacity to bind to the carbohydrate moiety.

Function of the cumulus oophorus before and during mammalian fertilization

Abstract: Contents Fertilization encompasses a series of different steps which have to be performed in a well-orchestrated way to create a new individual. They include sperm capacitation, sperm binding and penetration of the zona pellucida, traversing the perivitelline space, binding and fusion with the oolemma, activation of the oocyte and decondensation of the sperm head to form the male pronucleus. In most mammalian species, cumulus cells surround the oocyte at the time of fertilization. Removal of the cumulus oophorus at this point of time often leads to a drop in fertilization rates. It is not yet known how cumulus cells interact with the oocyte or with spermatozoa to promote fertilization. There are different possibilities: 1cumulus cells cause mechanical entrapment of spermatozoa and guide hyperactivated spermatozoa towards the oocyte, while preventing abnormal spermatozoa to enter the cumulus matrix; 2cumulus cells create a micro-environment for the spermatozoa which favours their capacitation and penetration into the oocyte; 3cumulus cells prevent changes in the oocyte which are unfavourable for normal fertilization; these changes can be located in the zona pellucida or in the cytoplasm. In this review, studies in several species are listed to prove the importance of these three cumulus cell functions and the current lines of research are highlighted. Moreover, different ways to improve in vitro fertilization of bovine cumulus-denuded oocytes are discussed.

Comparative analysis of protein synthesis, transcription and cytoplasmic polyadenylation of mRNA during maturation of bovine oocytes in vitro.

Abstract: Contents In many species, large numbers of macromolecules are accumulated during oocyte growth. The messenger and ribosomal RNAs produced in these cells are far in excess of those necessary to support protein synthesis. Thus, the aim of this study was to elucidate the processes of translational regulation during meiotic maturation. The relationship between transcription, translation and polyadenylation of mRNA during in-vitro maturation (IVM) of bovine oocytes was investigated. The results presented here show that overall protein synthesis is stimulated during meiotic maturation (approximately three times) concomitantly with the onset of germinal vesicle breakdown after 6 to 10 h of IVM. However, in metaphase II, the incorporation of [35S]methionine into proteins showed only basal levels, as in the germinal vesicle (GV) stage. Furthermore, in the course of IVM, de-novo transcription strongly declines as determined by measuring the incorporation of [3H]uridine into RNA. In contrast to this finding, the incorporation of [3H]adenosine increased and showed a peak during the time interval from 6 to 10 h of IVM, parallel with the onset of germinal vesicle breakdown (GVBD) and translation. In the further course of maturation, only a moderate decrease of [3H]adenosine incorporation was observed. These results indicate that (i) translation increased at the time of GVBD; (ii) these processes were accompanied by polyadenylation of mRNA; and (iii) although transcription declines, polyadenylated mRNA is accumulated until metaphase II (as shown by poly(U)-hybridization), in which protein synthesis is low. The correlation of these processes is discussed here. A detailed knowledge of the biochemical and molecular processes which occur during oocyte maturation can be useful for the improvement of IVM conditions.

Production of buffalo (Bubalus bubalis) embryos in vitro: premises and promises.

Abstract: Techniques for in vitro production (IVP) of buffalo embryos adopting the procedures developed in cattle have received increasing interest in the recent times. A high oocyte maturation, fertilization and cleavage rate and a low rate of blastocyst yield and calving following transfer of in vitro produced buffalo embryos have been obtained. The efficiency of IVP in buffalo is much lower than that in cattle. Several problems need to be resolved before IVP technology can be used regularly in buffalo breeding. This review attempts to present an overview of the different techniques used in buffalo to produce transferable embryos in vitro, namely in vitro maturation and fertilization of immature oocytes and in vitro development of the resulting cleaved embryos to the blastocyst stage before transfer. The problems associated with IVP, the possible solutions and the new biotechniques linked to IVP are discussed.

New Strategies to Prevent Mastitis

Abstract: Bovine mastitis remains as the disease causing the biggest economic losses to the dairy industry, despite the intensive research and prevention measures at herd level carried out for decades. Antibiotics are widely used to combat mastitis, but focus should be shifted from treatment to more economical and efficient prevention. The bacteriological aetiology of mastitis has changed from contagious to environmental pathogens, which has reduced the efficacy of the traditional mastitis control strategies. Considerable progress in the understanding of epidemiology, immunology, diagnostics and pathogenesis of mastitis has been made. The modern molecular biological methods offer good possibilities for the research of the epidemiological and virulence aspects of bacteria, which may help in building-up specific mastitis control strategies for dairy herds. Studies on the host response and relationship between somatic cell count and susceptibility to mastitis offer tools for genetic improvement of dairy cows. Biotechnological approaches for mastitis prevention are in the developmental stage, but many problems are associated, e.g. with vaccination of dairy cows against mastitis. Different methods of immunomodulation for the prevention of mastitis have shown promise in experimental trials, but the evidence is not yet enough to support commercial applications. Improving nutrition, housing and environment of dairy cattle are still crucial in the prevention of mastitis, especially during the most susceptible period after parturition. New milking techniques including robotic milking may provide better possibility for proper milking and improved udder health. Mastitis control should be part of the herd health programme in the dairy herds. In this paper, results from recent research and proposals for new prevention strategies in the field of mastitis are reviewed.

Follicle wave growth in cattle

Abstract: Ovarian follicle growth in cattle culminates in the selection of a single dominant follicle which attains the ability for final maturation and ovulation once or twice during the luteal phase and at the end of the oestrous cycle, as well as during other reproductive states This review will describe in detail the first follicle wave of the cycle leading to selection of the first wave dominant follicle, indicating the specific gonadotrophin dependencies of cohort and dominant follicles, and relating follicle fate to steroidogenesis As a differential gonadotrophin response of growing antral follicles during the follies-stimulating hormone (FSH) decline may determine which follicle becomes selected, first wave follicles are also characterized in relation to intrafollicular growth factors, which may modify the gonadotrophin response, such as inhibins and members of the insulin-like growth factor (IGF) family Subsequently, the follicular control of the transient FSH rise and decline so crucial to dominant follicle selection will be discussed It is concluded that successful hormonal manipulation of follicle wave growth and dominant follicle selection will depend on our detailed understanding of the gonadotrophin requirements of differentiating wave follicles

Blood and Intrauterine Leukocyte Profile and Function in Dairy Cows that Spontaneously Recovered from Postpartum Endometritis

Abstract: The profile and function of blood and uterine leukocytes were evaluated in 14 dairy cows that spontaneously recovered from postpartum endometritis (mild, n=6 and heavy, n=8; general health not affected). From a minimum of 2 weeks before parturition until 6 weeks postpartum, blood samples were obtained twice weekly for leukocyte counts and leukogram determination and once weekly for flow cytometry assessment of polimorphonuclear neutrophils (PMN) phagocytic capacity and oxidative burst activity. Uterine fluid-stained smears, obtained twice weekly from parturition until fluid was present in the uterus, were used for determination of the percentage of PMN, of phagocytizing PMN (phago-PMN) and of the mean number of phagocyted bacteria per phagocytizing PMN (phagocytic index; PI). Uterine swabs were obtained twice weekly from parturition until 35 days postpartum for bacteriological examination. The time of endometritis diagnosis was similar in cows with mild or heavy endometritis but the latter cows had a significantly longer persistence of the infection and of the isolation of Gram-negative anaerobes from the uterus. However, the effect of group (mild versus heavy) was not significant for all the blood and uterine parameters analysed. The effect of sampling day (within group effect) was significant (p<0.01 to p<0.00001) for all parameters, except for the blood monocyte count and the blood PMN phagocytic capacity, in which only a tendency for significance was observed (p<0.1). The effect of the interaction group x sampling day was significant only for the blood monocyte count. The phago-PMN and the PI were significantly correlated (r=0.70, p<0.001). A significant correlation was also observed between the uterine fluid phago-PMN and the blood PMN oxidative burst activity (r=-0.41, p<0.05). At the spontaneous recovery, the blood PMN oxidative burst activity was significantly higher (p<0.05) and the percentage of intrauterine phago-PMN and the PI were significantly lower (p<0.001 and p<0.01, respectively) than at diagnosis of endometritis. These results suggest that a decrease in blood PMN oxidative burst activity until the first week postpartum could be associated with an increased susceptibility to early postpartum endometritis. The later increase in this parameter as well as the increase in the intrauterine fluid phago-PMN and PI, might favour the spontaneous resolution of endometritis.

Effect of swim up and Percoll treatment on viability and acrosome integrity of frozen-thawed bull spermatozoa.

Abstract: The aim of this study was to investigate the effect of the swim up and Percoll methods to select frozen-thawed bull spermatozoa with high quality membrane and acrosomal integrity and final concentration. Semen samples from six Holstein-Friesian bulls were examined. The whole experiment was repeated three times. Before and after both treatments, spermatozoa were subjected to a double-staining method and evaluated by brightfield light microscope using 40x dry, or 100x oil immersion objectives. The concentration of spermatozoa evaluated by haemocytometer was 8.8 x 10(7)/ml after thawing, and the percentage of live cells with intact acrosome was 45.8%. Both treatments significantly increased the proportion of live spermatozoa compared with no treatment, and the use of Percoll gradient resulted in a significantly higher percentage of living cells with an intact acrosome (88.2%) than the swim up method (69.4%). The concentration of spermatozoa after Percoll separation (9.3 x 10(6)/ml) was higher than that after the swim up method (5.8 x 10(6)/ml). These results indicate that spermatozoa with a higher viability and acrosome integrity can be obtained by Percoll separation than by the swim up method. Therefore the use of Percoll-treated spermatozoa in IVF systems can be more expedient.

Assessment of Different Sperm Quality Parameters to Predict in vitro Fertility of Bulls

Abstract: Frozen-thawed semen from six bulls with high (> 60%) and low (20-35%) in vitro fertility was used for studying the predictive value of simple sperm quality tests with respect to in vitro fertilization (IVF) outcome as assessed by pronucleus (PN) formation ability. Sperm quality parameters, such as sperm concentration, motility, progressive motility, live-dead sperm ratio, morphology, membrane integrity, mitochondrial activity and acrosomal status were analysed using both conventional and automatic techniques at three time points during the IVF process, namely after sperm thawing, Percoll differential gradient centrifugation and IVF. Associations between the sperm quality parameters before and after IVF, and PN formation ability were assessed by using linear regression analyses. The percentages of motility, progressive motility and normal morphology determined after sperm thawing, and the percentage of live spermatozoa assessed after Percoll preparation by using nigrosin-eosin (N-E) staining showed a good correlation with PN formation ability, but the regression parameters were borderline not significant. These parameters formed the most reliable basis for predicting IVF outcome. After IVF, the percentage of live spermatozoa determined by using N-E staining was the only sperm quality parameter showing a significant association with the PN formation ability of a given bull. This sperm quality test can be used as a non-invasive method to estimate the PN formation ability of oocytes which are further cultured to assess embryonic development.

Low dose insemination in the sow--a review.

Abstract: Contents Artificial insemination (AI) in pigs has been established for about four decades but ejaculates are still used insufficiently. Higher demand of semen for AI and new techniques that involve low sperm concentration require the optimization of insemination protocols. Based on the knowledge of the physiology of sperm transportation and events in the female genital tract prior to fertilization, new strategies are under development to minimize sperm losses. One goal is to deposit the semen into the uterine horn rather than into the proximal cervix. It was shown that the minimal number of spermatozoa necessary for surgical AI at the utero-tubal junction (UTJ) were at least 1 x 10 6 diluted in 0.5 ml of a special extender. Artificial insemination into the distal part of the uterine horn required about 1 × 10 7 million sperm in 20 ml of extender. Meanwhile, first insemination devices for non-surgical intrauterine AI are commercially available. Using similar sperm concentrations as for surgical AI, non-surgical uterine insemination did not differ significantly from control inseminations in terms of pregnancy rate and litter size. With respect to the fertilizing capacities of their ejaculates, boars have to be selected more strictly for sperm quality parameters as most of the compensatory effects of sperm cells disappear in maximally extended semen samples.

Comparison of the Ability of Three Radioimmunoassay to Detect Pregnancy-associated Glycoproteins in Bovine Plasma

Abstract: Contents Pregnancy-associated glycoproteins (PAGs) constitute a large family of glycoproteins that are synthesized in the superficial layer of the ruminant placenta according to a spatial and temporal expression pattern. When PAGs are released in the maternal blood they can be used for pregnancy diagnosis, pregnancy follow-up and for the monitoring of the trophoblastic function. Three different radioimmunoassay systems (RIA 1, RIA 2 and RIA 3) using antisera produced against PAG I67 (RIA 1), PAG55+62 (RIA 2) and PAG55+59 (RIA 3) were used in this investigation in order to measure the PAG concentration in plasma samples withdrawn from pregnant cows and heifers during different periods following artificial insemination (AI). These systems were able to detect PAG molecules in the maternal blood as early as 21 days after AI in different concentrations (RIA 1: 0.43 ± 0.24 ng/ml, mean ± SD; RIA 2: 0.48 ± 0.24 ng/ml; RIA 3: 0.64 ± 0.37 ng/ml). On days 32 and 42 RIA 2 (4.30 ± 1.32 ng/ml and 5.56 ± 1.95 ng/ml) and RIA 3 (4.17 ± 1.15 ng/ml and 5.60 ± 1.89 ng/ml) presented significantly (p < 0.0001) higher PAG concentrations than those of RIA 1 (2.43 ± 0.81 ng/ml and 4.01 ± 1.48 ng/ml), respectively. After day 21, significant correlations (p < 0.0001; r ≥ 0.929) were determined between the three systems. Additionally the three individual PAG profiles presented in this study showed that PAG molecules secreted in the maternal blood between 21 and 50 days after AI were better recognized by the RIA 2 and RIA 3 systems. This study clearly indicated that the ability of a RIA test to recognize PAG molecules in the maternal blood can be improved by carefully selecting the antiserum.

Vitamin A and beta-carotene levels in plasma, corpus luteum and follicular fluid of cyclic and pregnant cattle.

Abstract: This study was carried out to examine the relationship between the corpus luteum (CL) weight, CL and follicle diameters and progesterone, beta-carotene and vitamin A levels in reproductive organs of cattle obtained from the slaughterhouse. The beta-carotene and vitamin A levels were determined in plasma, CL and follicular fluid (FF) using a spectrophotometric method at different stages of the oestrous cycle (n=40) and at 3-6 months of pregnancy (n=10). The diameters of the CL and follicle were measured using ultrasonography. Plasma progesterone concentrations were determined by an enzyme immunoassay method. The vitamin A levels of the plasma, CL and FF were not related to each other. The highest plasma vitamin A levels were observed in the proestrus and oestrus, at which periods follicular activity dominates. The vitamin A levels in the CL and FF were negatively related to the weight and diameter of the CL and the diameter of follicle, respectively. In contrast to vitamin A, beta-carotene concentrations of plasma, CL and FF were significantly correlated with each other. The highest beta-carotene levels in the plasma, CL and FF were found during pregnancy when there is maximal luteal function, and the beta-carotene level of the CL was significantly correlated with the weight and diameter of CL. Furthermore, the intrafollicular beta-carotene level was negatively correlated with the follicle diameter. There was a positive correlation between plasma progesterone level and the weight and diameter of the CL, but a negative correlation between plasma progesterone level and follicle diameter. Moreover, plasma, FF and CL beta-carotene levels were positively correlated with plasma progesterone levels. This study revealed that beta-carotene levels in the plasma, CL and FF were influenced by the stage of the oestrous cycle or the pregnancy and were related to bovine luteal function without depending on vitamin A.

Effects of VEGF and bFGF on Proliferation and Production of Steroids and Nitric Oxide in Porcine Granulosa Cells

Abstract: Ovarian angiogenesis, which is currently considered to be of crucial importance in controlling the growth of developing follicles, is a physiological process driven by a variety of angiogenic factors. Among these, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have been recognized as key players in promoting cell growth and differentiation. Porcine granulosa cells from small ( 5 mm) follicles were seeded at different densities in DMEM:Ham's F12 (1:1) with or without different concentrations of VEGF or bFGF. After 48 h of culture, media were assayed for oestradiol (E2) 17beta, progesterone (P4), nitric oxide (NO) and VEGF levels; in addition, cell proliferation was evaluated by 3H-thymidine incorporation assay. Both bFGF and VEGF effects on E2 and P4 production by cultured granulosa cells resulted to be dependent on follicle size. The bFGF was always ineffective in modulating cell proliferation, while VEGF exerted an inhibitory effect on the proliferation in the small follicle group and a stimulatory one in the medium and large follicle groups. The bFGF consistently reduced NO levels in culture media. The VEGF appeared to be ineffective in modifying NO production in the small follicle group, while it was stimulatory in the medium follicle group and inhibitory in the large follicle group. Basal VEGF production was higher in cells from the large follicle as compared with the small and medium follicle groups, and it was unaffected by bFGF. These results suggest that VEGF plays a modulatory role in granulosa cell functional activity and it is possibly involved in the regulation of follicle growth; on the contrary, bFGF does not appear to represent a significant regulatory factor in our cellular model, except for an inhibitory action on the production of NO, whose anti-angiogenic properties need to be further substantiated.

Cryopreservation of cat semen in straws: comparison of five different freezing rates.

Abstract: Contents The first pregnancies in domestic cats were obtained using semen frozen in pellets (Platz et al. 1978). Other freezing methods, vials (Lengwinant and Blottner 1994) or straws (Pope et al. 1991; Hay and Goodrowe 1993), have also been used. Pelleted freezing has often been the standard method (Howard 1986). Opinions about the freezing method are discordant; the best method for Pope et al. (1991) was using straws; in fact, the post-thaw motility and the percentage of normal acrosomes were of 44 ± 4 and 62 ± 3%, respectively, with straws and 11 ± 3 and 26 ± 4%, respectively, with pellets. According to Wood et al. (1993), there are no differences between the two methods, with a motility of 66.2% and a percentage of normal acrosomes of 28.6% for the pellet method and a motility of 67.0% and a percentage of normal acrosomes of 27.4% for the straw container. However, these two authors used two different freezing protocols. A high concentration of glycerol (i.e. 8%, vol/vol) damages cat semen (Zambelli 1994; Nelson et al. 1999), because of his toxicity to spermatozoa (Graham 1996); while a concentration of 4% is suggested (Zambelli 1994). Fast green FCF Bengal pink staining is often used to evaluate the acrosomal morphology (Wood et al. 1993; Zambelli et al. 1993). As there are no studies on the influence of freezing rate on motility and on acrosomal morphology, the aim of this study was to test five freezing rates in order to verify which is the best for the cryopreservation of cat semen in straws.

Insemination of susceptible heifers with semen from a non-viraemic bull with persistent bovine virus diarrhoea virus infection localized in the testes.

Abstract: Contents Bulls shedding bovine viral diarrhoea virus (BVDV) in semen and simultaneously having a high concentration of circulating antibodies may cause reproductive problems and spread the viral infection within cattle populations. To investigate this in detail, three heifers were inseminated with BVDV-infected semen from a non-viraemic, seropositive Holstein–Friesian bull, named `Cumulus'. One control heifer was inseminated with semen from a healthy bull that was free of BVDV. All four heifers remained clinically healthy throughout the experiment. The conception succeeded in the control animal and in two of the three heifers inseminated with semen containing BVDV. The heifer with the failed conception was the only one that became systemically infected with BVDV. This animal was deemed non-pregnant by ultrasonic examination on day 34 after insemination and showed no signs of subsequent oestrus during the entire experimental period. At slaughter, 42 days after insemination, there were no histopathological changes in the ovaries and virus was not detected in ovarian tissue. The fact that seronegative dams served with semen from persistently infected bulls have occasionally produced persistently infected calves together with the present findings and the fact that non-viraemic, seropositive bulls can constantly shed BVDV, suggest that the use of semen from such bulls in BVDV-free herds could have far-reaching consequences, especially if it led to the birth of persistently infected (P1) calves.

Vital aspects of Fallopian tube physiology in pigs.

Abstract: Contents This essay reviews four topical aspects of Fallopian tube physiology that bear on either successful fertilization or early development of the zygote. An initial focus is on glycoprotein secretions of the duct that accumulate as a viscous mucus in the caudal isthmus. Because this is the site of the pre-ovulatory sperm reservoir, an involvement of the secretions is considered in: preventing uterine and ampullary tubal fluids from entering the functional sperm reservoir; removing residual male secretions from the sperm surface; deflecting spermatozoa towards endosalpingeal organelles and reducing flagellar beat before ovulation. The subtle prompting of flagellar movement with impending ovulation is examined in terms of potential reactivation mechanisms, with overall control attributed to increasing secretion of progesterone. The site of full capacitation and the acrosome reaction in a fertilizing spermatozoon is then debated, with strong arguments pointing to completion of these processes in the specific fluids at the ampullary-isthmic junction. Finally, the synthetic activity of cumulus cells released at ovulation as a paracrine tissue in the Fallopian tube is highlighted with reference to steroid hormones, peptides and cytokines. Not only does the suspension of granulosa-derived cells influence the process of fertilization, but also it may amplify oocyte or embryonic signals to the endosalpinx and ipsilateral ovary.

An overview of low dose insemination in the mare.

Abstract: Contents The need for relatively high numbers of spermatozoa for artificial insemination limits our application of recently available technologies such as sex-sorted semen. The fertility of two different methods of low dose insemination using fresh, frozen and sex-sorted semen are compared in this overview. Satisfactory conception rates are described using very low doses of spermatozoa inseminated by either hysteroscopic or deep uterine insemination methods, proving the stallion is fully fertile. The hysteroscopic method appears to give higher conception rates when inseminating fewer than 5 × 10 6 spermatozoa and is therefore, the preferred method of insemination for sex-sorted spermatozoa. However, hysteroscopic deposition of low numbers of spermatozoa from infertile stallions does not appear to improve their fertility.

Correlation between the cell number and diameter in bovine embryos produced in vitro.

Abstract: This study was designed to examine the effects of age and developmental stage of in vitro-produced bovine embryos on the cell number of the embryos and to investigate the correlation between the cell number and diameter in the embryos. The diameter and cell number in blastocysts and expanded blastocysts collected on days 7-9 after in vitro fertilization (IVF) were examined. Although the diameters of the blastocysts collected on days 7 and 8 after IVF were smaller than those of the expanded blastocysts collected on day 9, the cell number in both types of embryos was similar. The cell numbers of the blastocysts and expanded blastocysts decreased with increasing embryo age. There were positive correlations between the cell number and diameter in bovine embryos at each stage collected on each day after IVF. However, the value of the correlation coefficient in the day-9 expanded blastocyst group tended to be higher than that in the other groups. These results indicate that the cell number of in vitro-produced embryos is affected by the embryonic stage and age. The diameter of the embryo may be potentially used for the viability testing of the expanded blastocysts collected on day 9 after IVF.

Maturation Status, Protein Synthesis and Developmental Competence of Oocytes Derived from Lambs and Ewes

Abstract: The efficacy of oocyte selection for in vitro embryo production depends on the abundance and diameter of follicles, cumulus layers around the oocytes and subsequent fertilization. Application of 'ovum pick-up' technique allows us to utilize partially matured oocytes for embryo production even from juvenile subjects. To compare their developmental competence, oocytes derived from lambs and ewes and cultured in maturation medium for up to 26 h were assessed at 2 h intervals by confocal microscopy after chromatin and microtubulin-specific fluorochrome labelling. Lamb oocytes reached second meiotic metaphase (MII) at lower numbers at 24 h (60.0%) and 26 h (28.6%) whereas 85.7% of adult-derived oocytes attained MII status by 24 h of maturation. Radiolabelling of oocyte proteins revealed higher incorporation of [(35)S-]-methionine and [(35)S]-cysteine in adult-derived oocytes compared to lamb oocytes. Although the cleavage rate of lamb oocytes was similar to that of ewe oocytes, the proportion reaching blastocyst stage was significantly lower (p < 0.05) in the lamb-derived oocytes. However, blastocysts from both types of oocytes displayed similar cell lineage allocations to inner cell mass and trophectoderm.

Seroprevalence of Canine Herpesvirus-1 in the Belgian Dog Population in 2000

Abstract: Canine herpesvirus-1 (CHV-1) is known to be associated with fertility and fecundity disorders as well as neonatal mortality in puppies of less than 3 weeks of age. The virus is presumed to be enzootic in dogs all over the world and recent studies in several European countries suggest a high seroprevalence among the dog population. In the year 2000, a total of 647 Belgian canine sera from 102 privately owned patients and 545 breeding dogs were analysed with an enzyme-linked immunosorbent assay (ELISA). Furthermore 77 of the samples were submitted to two serum neutralization (SN) tests for comparison. An overall CHV-1 seroprevalence of 45.75% was observed in the Belgian dog population. No significant differences could be observed based on breeding status, reason for consultation or sex. The correlation between the ELISA and both SN tests appeared to be moderate with a significantly greater sensitivity of the ELISA. This study also demonstrated that the CHV-1 seroprevalence in the Belgian dog population is similar to that in other recently investigated European countries and that the incidence in breeding units is not necessarily higher than in non-breeding dogs.

Reduced Phagocytotic Activity of Macrophages in the Bovine Retained Placenta

Abstract: This study was carried out to investigate the distribution of immune cells in the bovine placenta during the postpartum period and to compare these cells between normal and retained placenta, Within 1 h after normal calving, biopsy samples of placentomes were collected from 10 cows. The occurrence of retention of fetal membranes was monitored for more than 8 h post-calving, and the samples obtained were divided into two groups: normally discharged and retained placenta (n = 5 each). Immunohistochemical procedures were utilized to detect macrophages and T lymphocytes. Numerous CD14-positive macrophages were found in the stroma of both normal placenta and retained placenta whereas only a few CD3-positive T lymphocytes were found in both cases. However, histochemical staining for acid phosphatase, a predominant lysozomal enzyme, revealed that almost all macrophages showed strong enzyme activity in the normally discharged placentas, whereas in retained placenta the activity of acid phosphatase was conspicuously decreased in intensity. These results indicate that there are functional differences in placental macrophages between normal and retained placenta.

Embryonic signals and survival.

Abstract: The objective of this review is to give an overview of the signaling mechanisms between the conceptus and the mother before implantation. The interactions between the embryo and uterus are complex and essential for normal embryo development and implantation. Problems in the signaling mechanisms are thought to play a significant role in early embryonic mortality since a high rate of embryonic morality occurs during this period. This review will focus on the mechanisms involved in the development of the conceptus and the prevention of luteolysis. It is based primarily on what is known in ruminants but also refers to work in other species such as the mouse and primates.

Aspartic proteinase members secreted by the ruminant placenta: specificity of three radioimmunoassay systems for the measurement of pregnancy-associated glycoproteins.

Abstract: Pregnancy-associated glycoproteins (PAGs) isolated from the placenta of various ruminant species are enzymatically inactive members of the aspartic proteinase family. The measurement of these proteins in the maternal blood can be a good indicator of the presence of a live embryo. As certain aspartic proteinases are present in biological fluids in physiological and pathological conditions at various concentrations, it was necessary to determine the specificity of three radioimmunoassay (RIA) systems currently used for the detection of PAG molecules. Commercially available members of the aspartic proteinase family like pepsinogen, pepsin, chymosin, rennet, cathepsin D and renin were tested in a wide concentration range (10 ng/ml - 1 mg/ml). Pepsinogen cross-reacted in RIA 1, RIA 2 and RIA 3 over 1 mg/ml, 50 microg/ml and 500 microg/ml concentrations, respectively. In the presence of pepsin, cross-reaction was observed in RIA 1, RIA 2 and RIA 3 over 1 mg/ml, 500 microg/ml and 1 mg/ml concentrations, respectively. Chymosin and rennet could cross-react in RIA 2 and RIA 3, while renin and cathepsin D did not decrease the binding of the tracer to antisera more, than that of the minimal detection limit. As the plasma/serum concentrations of the examined aspartic proteinases reported in the literature were outside the concentration range where cross-reaction was observed, it can be concluded that these RIA systems were specific for the detection of PAGs in biological fluids.

Effect of feeding and body condition score on multiple ovulation and embryo production in zebu cows.

Abstract: The aim of this study was to standardize the feeding regimen and the body condition score (BCS) for maximum superovulatory responses in indigenous zebu cows. Ten regularly cycling 5-8-year-old dry cows, weighing 176--260 kg with BCS 2.5--4.5 were divided into two equal groups at random. The groups were maintained on either a good-nutrition or a high-nutrition diet. The feedstuffs were analysed by proximate feed analysis and the metabolizable energy content was estimated. After 3 months feeding, individual cows were injected (i.m) with 1500 IU pregnant mare's serum gonadotrophin (PMSG) at day 10 or day 11 of the oestrous cycle (day of oestrus = day 0). Alfaprostol (6 mg) was injected (i.m) 48 h after the injection of PMSG to induce oestrus. At day 6 or day 7 (day of insemination = day 1), the contents of individual uterine horns were flushed with 150-200 ml of phosphate-buffered saline + 0.2% bovine serum albumin using a two-way Foley catheter. The embryos were identified, evaluated and graded as excellent, good, fair or poor under a stereomicroscope. For the good- and high-nutrition diets, the daily intake of green grass, straw, concentrate, dry matter, crude protein and estimated metabolizable energy by individual cows were 5 and 6 kg, 3 and 3 kg, 1.5 and 3.5 kg, 4.87 and 6.82 kg, 0.39 and 0.74 kg, and 39.60 and 59.12 MJ, respectively. The protein content was 8 and 11% in the good- and high-nutrition diets, respectively. The two groups of cows on different nutritional diets differed significantly with regard to body weight, body condition score and number of palpated corpora lutea (p < 0.01). For cows on the good-nutrition diet, the median number of recovered embryos and transferable quality embryos were three and two, respectively. The recovery rate of embryos was 79.30% of palpated corpora lutea. Cows on the high-nutrition diet did not yield any embryos. The indigenous zebu cows fed on the good-nutrition diet with BCS 2.5-3 were considered suitable for the induction of superovulation, the cows on the high-nutrition diet with BCS 4-4.5 were unsatisfactory and were more prone to cyst formation in the ovaries.

Motility characteristics and fertilizing capacity of boar spermatozoa stained with Hoechst 33342.

Abstract: Flow cytometry sorting of spermatozoa using fluorescence dye Hoechst 33342 is the only effective sex selection methodology validated in numerous laboratories. This study was carried out to determine the effect of Hoechst 33342 on the motility and fertility of stained boar spermatozoa. Experiment 1 evaluated motility parameters (percentage of motile spermatozoa, velocity, angularity and oscillation) of boar spermatozoa stained with Hoechst 33342 by a computer-aided sperm analysis (CASA) instrument. Spermatozoa (30 million/ml) were divided into five treatment groups and stained during 1 h at 35 degrees C with 9, 18, 27, 60 and 90 microM of H33342. There were no differences in sperm motility patterns nor percentages of motile spermatozoa incubated in the presence of 9, 18 or 27 microM. Percentage of motile spermatozoa and motility parameters decreased significantly (p < 0.05) at 60 microM of Hoechst 33342. Spermatozoa were immotile at concentration of 90 microM. In experiment 2, pregnancy rates, farrowing rates and litter size from sows (n = 275) artificially inseminated (AI) with either Hoechst 33342 stained (27 microM) or unstained (control) spermatozoa were determined. Sows inseminated with stained spermatozoa had no significant lower pregnancy rate (88.33%) as compared with controls (90.32%). Staining neither affected farrowing rates (85.0 vs 87.7%) nor total number of piglets born (10.56 +/- 0.32 vs 10.47 +/- 0.24, stained and controls, respectively). No phenotypical abnormalities were registered among the newborn piglets. The data suggest that incubating spermatozoa with Hoechst 33342 at levels required for X- and Y-bearing chromosome sperm sorting, does not impair sperm viability or their fertility after AI.

The Use of HOS Test to Evaluate Membrane Functionality of Boar Sperm Capacitated in vitro

Abstract: The functional and structural integrity of sperm membrane are crucial for the viability of spermatozoa. The commonly used staining test (eosin + nigrosin) for assessing sperm membrane measures only its structural integrity. The hypoosmotic swelling test (HOS) originally developed for human sperm (Jeyendran et al. 1984) has been also applied to several species of domestic animals (bull, pig, horse, dog). The test enables to evaluate the functional status of the sperm membrane. The principle of HOS is based on water transport across the sperm tail membrane under hypoosmotic conditions. It has previously been used to assess the semen quality (Revell and Mrode 1994), to analyse fertilizing capacity (Rota et al. 2000; Perez-Llano et al. 2001) and also to detect viable, immotile cells for ICSI (Intra-cytoplasmic sperm injection) in human (Zeyneloglu et al. 2000). There are two procedures commonly used for sperm capacitation in the pig-sperm washing and incubation before insemination (Nagai 1994). Capacitation involves several changes like removing molecules coating the sperm head membrane, changes in membrane fluidity and intracellular ion concentration (Green and Watson 2001). Thus the membrane integrity as well as functionality may be affected as shown by Harrison (1996). The aim of the present study was to analyse changes in sperm membrane integrity after in vitro capacitation by use of the HOS test.

Investigation of cervical patency and uterine appearance in domestic cats by fluoroscopy and scintigraphy.

Abstract: Contents The cervical patency of six domestic female cats was monitored under sedation by infusion of contrast medium (Omnipaque) into the cranial vagina during early oestrus, mid-oestrus, late oestrus and interoestrus or a radiopharmaceutical (99mTc-HSA) during mid- and interoestrus in a non-ovulatory oestrous cycle. The transport of the contrast medium or the radiopharmaceutical through the cervix and within the uterine horns was observed under fluoroscopy and with the aid of scintigraphy. In three of the queens, transcervical transport of contrast medium was demonstrated in all stages of oestrus, in one queen during mid-oestrus, late oestrus and 1 day after oestrus, and in two queens only during late oestrus. The relations between the cervical patency to the contrast medium and the oestrous behaviour, cornification of the vaginal cells and the serum oestradiol-17β concentration were evaluated, and a relationship was found between the cervical patency and the degree of vaginal cornification. Transcervical transport of the radiopharmaceutical was observed in three queens during mid-oestrus. When the cervix was open, hysterography under a fluoroscope and hysteroscintigraphy were performed. The fluoroscopic and scintigraphic recordings revealed the patterns of the uterine contractions during oestrus in both ascending and descending directions, and the movement of the uterine contents back and forth between the uterine horns. The hysterograms were classified according to the shape of the uterine horns and the appearance of the endometrial lining. Spiral-shaped uterine horns with a smooth inner contour were observed in two queens, and a corkscrew appearance with irregular filling defects in the uterine lumen was shown in two queens that had developed subclinical cystic endometrial hyperplasia. These findings demonstrated that fluids or particles deposited in the cranial vagina of the cat can be transported into the uterus during some stages of the oestrous cycle. The fluoroscopic and scintigraphic techniques developed in this study may be further modified to permit more detailed studies of uterine contractile patterns and sperm transport in the feline female reproductive tract. Hysterography proved useful to diagnose uterine disease. The information on cervical patency is of value also for the development of techniques for artificial insemination in this species, and should be studied also in the ovulatory cycle.

Validation of the FACSCount AF system for determination of sperm concentration in boar semen.

Abstract: A flow cytometric method has been developed for rapid determination of sperm concentration in semen from various mammalian species.* All cells containing DNA are stained with SYBR-14 or propidium iodide (PI) and sperm concentration is determined in relation to an internal standard of fluorescent microspheres ( beads). Satisfactory staining can be achieved within 2-3 min and the following flow cytometric analysis on the FACSCount AF System rapidly provides the user with a precise and accurate assessment of the sperm concentration. In this study, the FACSCount AF System and Sperm Counting Reagent ( BD Biosciences) was compared with microscopic counting using a Burker-Turk haemocytometer. In addition, sperm concentration was determined using the Corning 254 spectrophotometer which is used routinely by Danish artificial insemination stations for boars. The results show that the agreement between flow cytometry and microscopic counting is very high. The slope for the regression line was 1.12 (SE = 0.03) with an estimated intercept with the Y-axis of 22 x 10(6) sperm/ml (SE = 10 x 10(6) sperm/ml) and an estimated error of the model of 10 x 10(6) sperm/ml. For the spectrophotometer, the slope of the regression line was 1.09 (SE = 0.07) with an estimated intercept of 137 x 10(6) sperm/ml (SE = 25 x 10(6) sperm/ml). The average error made by the spectrophotometer was 55 x 10(6) sperm/ml. In addition, the results obtained using flow cytometry was highly repeatable ( CV = 2.7%) in comparison with the spectrophotometric method ( CV = 6.3%). These results indicate that the FACSCount AF System is a valuable tool for precise and accurate assessment of sperm concentration in boar semen and that use of this system may lead to production of more uniform insemination doses containing a specific number of sperm per dose.

15‐Ketodihydro‐PGF2α, Progesterone and Uterine Involution in Primiparous Cows with Induced Retained Placenta and Post‐partal Endometritis Treated with Oxytetracycline and Flunixin

Abstract: Retention of the foetal membranes (RFM) and post-partal endometritis are common problems in dairy cows. Among other things, the disease is characterized by a bacterial endometritis with aerobic as well as anaerobic bacteria. From an endocrine perspective, cows with RFM have high levels of 15-ketodihydro-PGF(2 alpha) (PG-metabolite) immediately after parturition but these levels fall rapidly within 2 weeks post-partum (early PG-metabolite elevation). After this decline, the PG-metabolite levels increase again and the levels (at this time of a lower magnitude) remain elevated during the period of uterine infection (late PG-metabolite elevation). The aim of this study was to investigate the PG-metabolite profiles in cows with retained placenta and post-partal endometritis treated with the prostaglandin synthesis inhibitor flunixin (F), either alone or in combination with oxytetracycline (T). The study was accomplished over 2 years with 12 primiparous cows in each experiment. As a model for RFM, preterm parturition was induced in late-pregnant heifers by injecting PGF(2 alpha) (25 mg i.m) twice with a 24 h interval. In each experiment, the cows were divided into four groups and treated with either T (10 mg/kg b.w. i.m. once daily), F (2.2 mg/kg b.w. p.o. twice per day), a combination of T and F (dosage, as above), or conservatively (0). The treatment periods lasted from day 11 to day 14 post-partum (pp) in experiment 1 (after placental shedding, groups T1, F1, TF1 and 0) and from day 3 to day 6 pp in experiment 2 (before placental shedding, groups T2, F2, TF2 and 0). Jugular vein blood samples were collected for analyses of PG-metabolite and flunixin. Uterine biopsies were collected twice weekly for investigation of endometrial microbiology. Rectal palpation and ultrasonographic examinations were performed three times per week for investigations of uterine and cervical involution and ovarian activity. No attempts were made to remove the placentas manually. The experiment lasted until day 56 pp. The induction of parturition was successful in all heifers and 22 of 24 animals had RFM. All RFM cows had bacterial endometritis, based on bacteriological examinations. Flunixin treatment (F1, TF1, F2 and TF2) suppressed PG-metabolite levels significantly (p=0.006) during the period of treatment in both experiments. However, the early flunixin treatment only suppressed PG synthesis partially. Late oxytetracycline treatment (T1) did not influence the PG-metabolite levels but oxytetracycline treatment (T2 and TF2) before placental shedding significantly altered the kinetics of the early PG-metabolite elevation compared with other treatments. Late PG-metabolite elevation was significantly correlated to duration of uterine infection and cervical involution. In conclusion, flunixin treatment of cows with retained placenta either before or after placental shedding suppresses prostaglandin synthesis. However, early treatment, when the release of prostaglandins is high, might need more intensive treatment in order to prevent the PG synthesis effectively. Oxytetracycline treatment during the period immediately after parturition before placental shedding might influence the PG-metabolite profile and suggests a bacteriological contribution to the high levels of PG-metabolite seen during the first 2 weeks pp in cows with retained placenta. In this study, a correlation between prostaglandin release, the final cervical involution and the end of infection was found. This suggests a link between uterine endocrinology, bacteriology and involution in cows with retained placenta.