On the Species Specificity of Sperm Binding and Sperm Penetration of the Zona Pellucida

Sperm binding and sperm penetration of the zona pellucida (zp) are regarded as species‐specific. In this investigation, the interactions between bovine oocytes and porcine, respectively, equine spermatozoa have been studied under in vitro conditions and compared with the normal in vitro fertilization of bovine oocytes by bovine sperm. Surprisingly, many of the heterologous spermatozoa adhered firmly to the bovine oocytes and could not be removed by intense washing. On average, more than 100 boar or equine spermatozoa were bound to the zp of bovine oocytes. Electron microscopic studies clearly demonstrated that porcine sperm attached to the zona and underwent the acrosome reaction. Equine spermatozoa displayed a similar binding affinity, but unlike the porcine spermatozoa even penetrated the zp and were taken up into the oocyte after a longer period of co‐incubation. Considering these new results the dogma of a strict species specificity of sperm zona interactions under in vitro conditions has to be reconsidered.     

Effect of Protease Inhibitors on the Acrosome Reaction and Sperm‐Zona Pellucida Binding in Bovine Sperm

Acrosomal proteases participate in several events during fertilization process and are necessary during the acrosome reaction (AR) and sperm‐zona pellucida (ZP) binding process. In this study, the participation of sperm trypsin‐like, chymotrypsin‐like, and metalloprotease enzymes in the AR and ZP binding in cattle was investigated using protease inhibitors. Motile bovine sperm were obtained by a swim‐up method (4 × 10⁶ cells / ml) in Brackett and Oliphant medium. The sperm were capacitated and then incubated with Antithrombin III (trypsin and chymotrypsin inhibitor); N‐α‐p‐tosyl‐l ‐lysine‐chloromethyl‐ketone (trypsin inhibitor); Trypsin inhibitor (I‐S Type from soybean); N‐tosyl‐l ‐phenylalanine‐chloromethyl‐ketone (chymotrypsin inhibitor); or disodium salt from the hydrated ethylenediaminetetraacetic acid (metalloprotease inhibitor). Then, the AR was induced with lysophosphatidylcholine and evaluated with the double stain technique. Sperm‐zona binding capacity was evaluated using cumulus cell‐free oocytes. A significant decrease (p < 0.05) in the percent of true acrosome‐reacted sperm was observed only in cells incubated with trypsin (10.2 ± 1%) and chymotrypsin inhibitors (18.5 ± 1%) in relation to the control (52.2 ± 1%). Treatment with the metalloprotease inhibitor did not affect the AR percentage (51.8 ± 1%). On the contrary, there was no significant change in the number of sperm bound to the ZP with any of the inhibitors used. The results suggest a role for trypsin and chymotrypsin proteases, but not metalloproteases, in the AR in bovine sperm. In addition, these proteases do not seem to be involved in the binding of bovine sperm to the ZP.     

In vitro Effect of Zearalenone and α-Zearalenol on Boar Sperm Characteristics and Acrosome Reaction

This study was conducted to determine the in vitro effects of three different concentrations (125, 187.5 and 250 microM in diluted semen) of zearalenone (zen) and alpha-zearalenol (alpha-zen) on boar sperm. Semen parameters such as motility, viability and spontaneous acrosome reaction were evaluated. From the results it was shown that both zen and alpha-zen affected the sperm characteristics significantly (p < 0.05), except for alpha-zen at the low concentration which did not decrease the percentage of live reacted spermatozoa significantly. In conclusion, zen and alpha-zen are directly toxic when they affect boar semen in vitro and consequently decrease the fertilization ability of the sperm. The higher the concentration of mycotoxin tested, the greater the decline of sperm parameters noticed. The influence of mycotoxins was found to be time- and dose-dependent.     

Inhibition of rumination in sheep by α-adrenoreceptor antagonists

Rumination activity was recorded in sheep during a period of 3 h. The amount of rumination occurring in 30 min following the intravenous injection of an alpha 1 blocking drug (prazosin, 0.1 mg/kg) and an alpha 2 blocking drug (idazoxan, 0.1 mg/kg) was reduced compared with control days when saline was given. The observation that rumination can be stimulated by a rapid, intravenous dose of adrenalin was confirmed. The induction of rumination by adrenalin was prevented by these alpha-adrenoreceptor antagonists given singly or together at the dose rate mentioned. These findings are discussed in the light of knowledge that normal reticulo-ruminal motility and food intake can be inhibited by stimulation of alpha 2-adrenoreceptors.     

Expression and localization of estrogen receptors α and β mRNA in medullary bone of laying hens

The aim of this study was to observe the expression and localization of estrogen receptor (ER) α and ER β mRNA in the medullary bone of laying hens. First, medullary bone, liver, kidney, and shell gland of the oviduct tissues were dissected from laying hens. Then, the total cellular RNA was isolated from each tissue specimen, and the ER α and ER β mRNA expression was observed using semiquantitative RT‐PCR. Second, the localization of ER α mRNA in the medullary bone was detected with in situ hybridization using digoxigenin‐11‐UTP‐labeled cRNA probes. As a result, the expression of ER α mRNA was higher than that of ER β mRNA in the medullary bone, liver, and shell gland of the oviduct from laying hens. In the kidney, ER α mRNA expression was lower than that of ER β mRNA. The expression pattern of ER α and ER β mRNA of the medullary bone was similar to that of the shell gland of the oviduct. Moreover, ER α mRNA was intensively expressed in osteoblasts on the medullary bone surface and bone marrow stromal cells but was not expressed in osteoclasts. These results suggest that in medullary bone, estrogen action may be regulated not by ER β but by ER α.     

Immunohistochemical Localization of Oestrogen Receptors α and β, Progesterone Receptor and Aromatase in the Equine Placenta

The functions of placental oestrogens during equine pregnancy are still unclear. Yet, they may act predominantly as local regulators of growth and differentiation in the microplacentomes. Thus, expression patterns of oestrogen receptors (ERs) α and β were investigated in the microcotyledonary placenta from pregnant mares at 110, 121, 179, 199 and 309 days of gestation by immunohistochemistry. In microplacentomes, both the ER isoforms were detected in trophoblast (T) cells, chorionic villous stroma (FS), microcaruncular epithelium (ME) and microcaruncular stroma (MS). Proportions of positive cells were 38–91% (T), 11–41% (FS), 55–89% (ME), 17–51% (MS) for ERα and 66–76% (T), 21–37% (FS), 41–68% (ME) and 24–55% (MS) for ERβ. Between days 110 and 199, proportions of cells positive for progesterone receptor (PR) varied between 19% and 62% (T), 3% and 50% (CS), 15% and 46% (ME), and 4% and 33% (MS). At day 309, PR was virtually absent in T, CS and ME (percentages < 0.1), whereas in MS 14.3% of cells were still positive. The expression of ERs and PR in equine microplacentomes gives evidence for a role of placental steroids as regulators of placental growth, differentiation and function. The detection of ERα, ERβ and PR in foetal and maternal vascular tissue suggests that placental steroids are also involved in the control of placental angiogenesis and /or vascular functions. The co-localization of ERs with aromatase in T suggests auto- or intracrine functions of oestrogens in this cell type.     

Methyl‐β‐Cyclodextrin Improves Sperm Capacitation Status Assessed by Flow Cytometry Analysis and Zona Pellucida‐Binding Ability of Frozen/Thawed Bovine Spermatozoa

Mammalian sperm undergo a series of biochemical transformations in the female reproductive tract that are collectively known as capacitation. Cyclodextrins added to the sperm culture medium have been described to induce in vitro sperm capacitation, enabling its use in protein‐free media. However, the additive capacitating effect of methyl‐β‐cyclodextrin (MβCD) in the medium containing bovine serum albumin (BSA) is unknown in the bovine species. In this study, we evaluated the effects of incubating frozen–thawed bovine spermatozoa in a BSA‐containing medium supplemented with MβCD on different sperm quality and functional parameters. Sperm viability decreased with the addition of MβCD in a dose‐dependent manner (p < 0.05), and DNA damage could be observed but only with the highest concentration of MβCD. However, pre‐incubation of spermatozoa in MβCD‐supplemented medium improved the capacitation status as assessed by the increase in plasma membrane fluidity, intracellular calcium concentration, induced acrosome reactivity and zona pellucida (ZP)‐binding ability (p < 0.05). Thus, we conclude that MβCD supplementation is able to enhance the capacitation status of frozen–thawed bovine spermatozoa cultured in capacitation medium containing BSA and could result in a valid strategy for its application on artificial reproductive technologies such as in vitro fertilization or intracytoplasmic sperm injection.     

Lactose Synthase Components in Milk: Concentrations of α-Lactalbumin and β1,4-Galactosyltransferase in Milk of Cows from Several Breeds at Various Stages of Lactation

It is believed that milk production is determined by the number and activity of mammary secretory cells. Secretory activity, as assessed by milk volume, depends on secretion of the major osmole in milk, lactose, which is produced by lactose synthase. The amount of either of the two proteins in lactose synthase may regulate milk production. The objective of this study was to determine whether the concentrations in milk of the two components of lactose synthase, α‐lactalbumin (α‐LA) and β1,4‐galactosyltransferase (B4GALT), were related to genetic background, stage of lactation, breed or parity of dairy cows. α‐Lactalbumin and B4GALT concentrations were measured by ELISA and by enzyme assays, respectively, from single milk samples. Two herds with a total of 279 cows were used in the analysis. One herd contained Ayrshire, Brown Swiss, Holstein and Jersey cows; the second herd contained two groups of cows; Holsteins selected for high milk production and Holsteins with 1960s genetics. The α‐LA concentration in milk was greater in Jerseys and Ayrshires than in Holsteins and Brown Swiss. However, no difference in α‐LA concentration was observed in milk from high and low genetic merit cows in the Minnesota herd or among different genetic backgrounds in the Illinois herd. β1,4‐Galactosyltransferase concentrations were similar for all groups that were analyzed. α‐Lactalbumin concentrations were positively correlated with milk protein concentration, milk fat concentration and lactose concentration. β1,4‐Galactosyltransferase concentration in milk exhibited a strong positive correlation with number of days in milk. Although the concentration of B4GALT increased as lactation progressed, the values did not show any correlation with persistency of lactation or late lactation milk production. In conclusion, this survey shows that the two components of lactose synthase are each correlated to protein concentration and individually correlated to the concentration of other milk components and stage of lactation.     

Ameliorative Effects of Melatonin against Hydrogen Peroxide‐Induced Oxidative Stress on Boar Sperm Characteristics and Subsequent In Vitro Embryo Development

Melatonin, the major secretory product of the pineal gland, scavenges a variety of reactive oxygen and nitrogen species in vivo and in vitro, indicating that melatonin is a potent function as an antioxidant. The objective of this study was to investigate the effect of melatonin in the presence or absence of hydrogen peroxide (H₂O₂) on sperm characteristics (motility, viability, survival rate, membrane integrity, lipid peroxidation (LPO) and mitochondria activity) and also to examine the developmental rates to the blastocysts stage of porcine oocytes fertilized in vitro with semen treated with or without melatonin (100 nm ) in the presence or absence of H₂O₂ (250 μm ). The sperm were treated with melatonin in the presence or absence of H₂O₂ for 3, 6, 9 and 12 h at 37°C and then analysed for the sperm characteristics. The porcine embryos were produced by in vitro maturation and in vitro fertilization (IVM/IVF) using semen treated with or without melatonin (100 nm ) in the presence or absence of H₂O₂ (250 μm ) for 6 h. The semen characteristics, including motility, viability, survival rate, membrane integrity and mitochondria activity, were higher in the groups that were treated with melatonin in comparison to other groups, irrespective of incubation periods. Malondialdehyde levels in control, melatonin and melatonin + H₂O₂ groups were lower than H₂O₂ only group. A positive correlation was shown among motility, viability, survival rate and membrane integrity, but a negative correlation was observed between LPO and the other evaluation methods. The developmental rates to blastocysts of IVM/IVF porcine oocytes fertilized by semen treated with melatonin were significantly increased compared with any other groups, with the cell number of blastocysts shown to have a similar trend to the developmental rates. These results demonstrate that melatonin can improve the semen characteristics during in vitro storage and support the developmental ability of IVM/IVF embryos in pigs.     

Molecular genetic characterization of ovine β-lactoglobulin C allele and detection by PCR-RFLP

Introduction β-Lactoglobulin (LGB), the major whey protein in sheep, consists of 162 amino acids and forms stable dimers in milk. Apart from its ability to bind and transport small hydrophobic molecules in milk, e.g. retinol and small fatty acids, its biological function is still unclear (Godovac -Zimmermann et al. 1985; Mac Leod et al. 1996). A positive effect on the digestion of milk lipids has been suggested (Perez et al. 1992). In nonruminant species two different forms, LGB I and II, are found, but in the milk of cattle, sheep and goat only one LGB is expressed. An LGB-pseudogene, lacking an active promotor was characterized by Passey and Mackinlay (1995). The deduced amino acid sequence is closely related to LGB II found in cat and horse, while the expressed LGB corresponds to LGB I. The LGB-pseudogene was mapped close to the LGB genes in cattle, goat and sheep (Passey and Mackinlay 1995; Folch et al. 1996). Three allelic variants (A, B and C) of ovine LGB can be discriminated using different electrophoretic techniques. Variants A and B are present in all breeds and differ by a Tyr/His substitution in position 20 (Kolde and Braunitzer , 1983) corresponding to a single nucleotide substitution in the LGB gene (Ali et al. 1990). A molecular genetic test for the differentiation of the A and B alleles by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) (RsaI) was developed (Schlee et al. 1993). The C variant was described first in 1989 in German Merinoland sheep at a frequency of 0.175 and in Hungarian Merino crosses (Erhardt 1989). In Spanish Merinos the C allele was found at low frequencies (0.013), too (Recio et al. 1995). There is little information available concerning the correlations of different LGB genetic variants with milk yield, protein content, cheese-making ability and other economic traits in sheep (Bolla et al. 1988; Garzon and Martinez 1992; Recio et al. 1995), but significant effects of LGB genotypes on whey and the resulting total protein content (Ng -Kwai -Hang et al. 1990; van den Berg et al. 1992; Hill 1993) and milk fat content (Aleandri et al. 1990; Falaki et al. 1997) were found in cattle. As the Merino breeds are one of the most widespread breed groups in the world, the inclusion of the LGB*C allele in further investigation of correlations of LGB genotypes with quantitative or economic traits in sheep is highly desirable. In addition, the occurrence of rare variants might help to clarify breed relationships. Examination of the LGB*C primary structure revealed a single amino acid substitution (Arg/Gln) in position 148 compared to the most common A allele (Erhardt et al. 1989). The test described by Schlee et al. (1993) does not discriminate LGB*C from LGB*A and leads to misidentification in animals with LGB*C. According to Ali et al. (1990) and Harris et al. (1993) amino acids 117–150 are encoded in exon V of the gene. Therefore this region was examined in the present study. Parts of exon V to VI of the gene were PCR-amplified, cloned and sequenced. Based on the sequencing results a PCR-RFLP typing procedure for identification of LGB*C was developed.     

Comparison of the effects of etomidate and its fluoro analogue, R 8110, on plasma Cortisol, 11β-deoxycortisol, 17α-hydroxyprogesterone and testosterone concentrations in dogs

R 8110, an imidazole derivative, was shown to be clinically superior to etomidate for induction and maintenance of anaesthesia in dogs. The present study compared the effects of intravenous (i.v.) R 8110, etomidate and Ringer solution on cortisol biosynthesis by the adrenal gland in seven male labradors. A tetracosactide challenge was carried out 30 min after the i.v. injection of 3 mg/kg of both drugs and after i.v. Ringer solution (1 ml/kg). Etomidate and R 8110 both suppressed the cortisol response to tetracosactide almost completely and increased the plasma 11 beta-deoxycortisol levels more than 20 fold. Maximal 11 beta-deoxycortisol values were reached 120 min after R 8110, and not less than 300 min after etomidate. Plasma 17 alpha-hydroxyprogesterone and testosterone concentrations did not differ between placebo and R 8110 treatment, but they decreased after etomidate. These results indicate that the effects of R 8110 on steroid biosynthesis in dogs are less pronounced than those of etomidate and are largely limited to a temporary inhibition of the 11 beta-hydroxylase in the adrenal gland.     

Regulation of uterine function by cytokines in cows: Possible actions of tumor necrosis factor-α, interleukin-1α and interferon-τ

When animals do not become pregnant, regression of the corpus luteum (CL) is essential for normal cyclicity because it allows the development of a new ovulatory follicle. Luteal regression is caused by a pulsatile release of prostaglandin (PG) F_2α from the uterus in the late luteal phase in most mammals including cattle. Although it has been proposed in ruminants that pulsatile PGF_2α secretion is generated by a positive feedback loop between luteal and/or hypophyseal oxytocin and uterine PGF_2α, the bovine endometrium may possess other mechanisms for initiation of luteolytic PGF_2α secretion. There is increasing evidence that several cytokines mainly produced by immune cells modulate CL and uterine function in many species. Tumor necrosis factor‐α (TNF‐α) stimulates PGF_2α output from bovine endometrium not only at the follicular phase but also at the late luteal phase. Administration of TNF‐α at a high concentration prolongs luteal lifespan, whereas administration of a low concentration of TNF‐α accelerates luteal regression in cows. The data obtained from the authors’ previous in vitro and in vivo studies strongly suggest that TNF‐α is a crucial factor in regulating luteolysis in cows. The authors’ recent study has shown that interleukin‐1α mediates PG secretion from bovine endometrium as a local regulator. Furthermore, interferon‐τ (IFN‐τ) suppresses the action of TNF‐α on PGF_2α synthesis by the bovine endometrium in vitro, suggesting that IFN‐τ plays a luteoprotective role by inhibiting TNF‐α‐induced PGF_2α production in early pregnancy. The purpose of the present review is to summarize current understanding of the endocrine mechanisms that regulate uterine function by cytokines during the estrous cycle and early pregnancy in cows.     

Role of Tumour Necrosis Factor α in Stimulation of Prostaglandins F2α and E2 Release by Cultured Porcine Endometrial Cells

The present studies were undertaken to examine the effect of tumour necrosis factor (TNF) alpha on prostaglandins (PGs) F(2alpha) and E(2) release by cultured porcine endometrial cells harvested on days 13-16 after oestrus in comparison to stimulation with oxytocin (OT) and luteinizing hormone (LH). A time-dependent effect of TNFalpha (10 ng/ml) on PGF(2alpha) release was observed in stromal and luminal epithelial cells. Moreover, TNFalpha increased PGF(2alpha) secretion from both endometrial cell types with effective concentrations of 1 (p < 0.05), 10 and 50 ng/ml (p < 0.01). The effect of TNFalpha (10 ng/ml) on endometrial PGF(2alpha) and PGE(2) release was compared with OT (100 nmol/l) and LH (100 ng/ml). All factors affected PGF(2alpha) secretion from stromal cells, however, the stimulation tended to be more potent after OT and LH (p < 0.01) than after TNFalpha (p < 0.05) treatment. In epithelial cells, only TNFalpha was able to stimulate PGF(2alpha) release (p < 0.001). PGE(2) secretion from stromal cells increased after incubation with TNFalpha and OT (p < 0.05). Only LH stimulated PGE(2) release from epithelium (p < 0.001), and its action was very effective when compared with TNFalpha or OT (p < 0.01). Summarizing, TNFalpha induces both PGs secretion from cultured porcine endometrium, but preferentially stimulates PGF(2alpha) release from luminal epithelial cells. Therefore, similarly to OT and LH, TNFalpha may be considered as a potential modulator of endometrial PGF(2alpha) production during luteolysis in the pig.     

OC1Effects of Cryopreservation on the Expression of Glut‐3, Glut‐5 and As‐A Proteins in Iberian Boar Sperm Membranes

In order to determine the injure produced in boar spermatozoa through cryopreservation process, we analyzed the expression of the hexose transporters Glut‐3 and Glut‐5 and the zona pellucida binding protein As‐A (P68) in three different steps of the freezing‐thawed protocol: at 17°C (fresh BTS‐diluted semen, 1 : 2 v/v, step 1), at 5°C (after glycerol addition; step 2), and post‐thawing (step 3). All sperm analyses were carried out with immunogold techniques under electronic microscopy. For this study eight healthy post‐pubertal Iberian boars were submitted to a collection of twice per week through 3 months, evaluating two ejaculates from each boar. Glut‐3 maintains the expression in the acrosome region post‐thawing but not along the tail where is reduced. The expression of Glut‐5 and As‐A is majority located at the post‐acrosome region of the spermatozoa at step 1, but in step 2 and step 3 this expression is relocated to sperm tail area. In conclusion, while cryopreservation affects the localization and the expression of Glut‐3 and Glut‐5, its fertilizing capacity is not significantly reduced. The stabilization of boar semen at 5°C was found to be the most crucial step for sperm survival.     

Inhibition of bovine α-glucosidase by Castanospermum australe and its effect on the biochemical identification of heterozygotes for generalised glycogenosis type II (Pompe''s disease) in cattle

All 18 2-year-old Brahman bulls grazing in a paddock containing Castanospermum australe trees were diagnosed as heterozygotes for Pompe's disease by measurement of mononuclear cell alpha-glucosidase activity. However, removal of the bulls to a paddock free of C. australe and retesting 2 months later indicated that 15 were homozygous normal. An in vitro assay demonstrated that a crude aqueous extract of seeds from these C. australe trees contained a potent inhibitor of mononuclear cell alpha-glucosidase. Two Hereford steers were dosed with 0.6 g C. australe seed/kg bodyweight for 6 days. The alpha-glucosidase activity in blood mononuclear cells declined to 5% of normal within 48 h of commencement of dosing. It was therefore assumed that the bulls had consumed C. australe seeds. A means of differentiating true heterozygotes from animals consuming the toxic seed, using the ratio of plasma alpha-glucosidase activity at pH 5.6 to that at pH 3.7, is proposed.     

Hormonal Response of Female Goats to Active Immunization against a Recombinant Human Inhibin α-subunit, and establishment of an Enzyme-linked Immunosorbent Assay for Caprine Follicle-stimulating Hormone

The effect of selective immunosuppression of endogenous inhibin in goats on FSH, LH, progesterone and estradiol-17β profiles was studied during the breeding and nonbreeding seasons. Eighteen adult female Boer goats were immunized against the recombinant human inhibin α-subunit (hINH-α). With the exception of estradiol, which was determined by radio-immunoassay (RIA), all plasma hormone concentrations were determined by ELISA. The ELISA for FSH presented in this paper was established in the authors' laboratory, based on an existing RIA. Mean basal concentrations of FSH were not affected by immunosuppression of endogenous inhibin, nor was there a difference in the amplitude of the pre-ovulatory FSH surge. Immunization against inhibin appears to eliminate the slight secondary rise of FSH occurring 12–20 h after the major surge associated with ovulation. The LH profiles of the immunized goats were characterized by lower basal concentrations both before and after the pre-ovulatory LH surge which itself was reduced by 50% in immunized does. By contrast, concentrations of circulating estradiol were significantly elevated after inhibin-immunization. Progesterone profiles were not affected. Extending immunization into the anoestrous season by a booster injection of hINH-α, implicating oestrus induction with a progestagen and eCG, produced no discernible differences in FSH and LH profiles in comparison with nonimmunized control goats. The findings suggest that in goats, paracrine factors may play a more significant role in controlling follicular activity than a feedback mechanism acting via the pituitary.