Changes in phospholipids, cholesterol and protein content of oviduct fluid of cows during the oestrous cycle

The oviducts of 4 cows were cannulated and oviduct fluid was collected daily from the exteriorized cannulas for a total of 5 oestrous cycles. Daily serum samples were assayed for oestradiol-17 beta and progesterone to monitor the oestrous cycle. Data for each cycle were compared for oviduct fluid collected during the non-luteal phase (serum progesterone less than or equal to 1.5 ng/ml) and the luteal phase (serum progesterone greater than 1.5 ng/ml). During the non-luteal phase oviduct fluid volume was higher and the osmolality was lower than during the luteal phase. Total protein, cholesterol and phospholipid secreted daily was greater during the non-luteal phase. Cholesterol and protein concentrations were generally lower during the non-luteal phase, but phospholipid concentrations were generally higher. About 40% of the phospholipid in oviduct fluid was phosphatidylcholine and lysophosphatidylcholine, while phosphatidylinositol and lysophosphatidylinositol accounted for 20%. The ratio of 1-acyl-phospholipid to diacylphospholipid increased during the non-luteal phase. An increased cholesterol to phospholipid ratio, and a decreased cholesterol to protein ratio in oviduct fluid also were associated with the non-luteal phase. Changes in the lipid composition of oviduct fluid during the oestrous cycle may play a role in the preparation of gametes for fertilization.     

Oestradiol and progesterone receptors in the pig oviduct during the oestrous cycle

The concentrations of the tissue receptors for oestradiol (E) and progesterone (P) in the porcine oviduct at different stages on the oestrous cycle have been investigated by in vitro binding and exchange methods. Both hormones bound to specific cytoplasmic (Rc) and nuclear (Rn) receptor proteins with high affinity. The concentrations of ERc and ERn were two-fold higher in the ampulla as compared to the isthmus. The amount of ERc in the isthmic portion of the oviduct did not vary throughout the oestrous cycle. However, the ampullar ERc concentrations increased during prooestrus, showed a maximum at standing oestrus, thereafter decreasing. Significant variations in the amount of oviductal ERn were observed. Despite the differences in ERn amounts between segments, the concentration of ERn increased significantly during late prooestrus, attaining a three-fold elevation and remaining elevated during the period of standing oestrous and early luteal phase (days 3-4), thereafter returning to basal levels. No significant variations in the amount of isthmic PRc were found throughout the period studied. The ampulla, however, showed a significant increase in PRc concentrations during standing oestrus, thereafter decreasing. The concentrations of PRn in isthmus and ampulla were of about the same magnitude and varied significantly during the oestrous cycle, increasing in concentration from standing oestrous onwards. The temporal relationships between the variations in levels of oestradiol and progesterone receptors in oviductal tissues and those of the circulating plasma levels were established. The data obtained in this study suggest a relationship between the changes in the levels of oestradiol and progesterone oviductal binding during the first days of the oestrous cycle, and the gamete and embryo transport throughout the oviduct in the porcine species.     

Influence of the breed of bull (Bos taurus indicus vs. Bos taurus taurus) and the breed of cow (Bos taurus indicus, Bos taurus taurus and crossbred) on the resistance of bovine embryos to heat

In vitro studies have shown that Bos taurus indicus (B. t. indicus) embryos submitted to heat shock at early stages of development are better able to survive as compared to Bos taurus taurus embryos. Embryo genotype influences resistance to heat shock thus leading to the question as to whether embryos sired by thermo-tolerant breeds exhibit the same resistance to heat shock. In the present study the influence of both oocyte and semen, on the resistance to heat shock (HS) at early stages of in vitro development, was assessed in B. t. indicus [Nelore (N) breed], B. t. taurus [Holstein (H) and Angus (A) breeds] and crossbreds. In Experiment 1, Nelore and crossbred oocytes were collected from slaughterhouse ovaries and fertilized with spermatozoa from Nelore and Angus bulls. Presumptive embryos were collected and randomly assigned to control (39 degrees C) or HS at 12, 48 or 96 h post insemination (hpi; 41 degrees C for 12h) treatments. The cleavage rates and proportion of embryos developing to the blastocyst and hatched blastocyst stages were recorded on Days 2, 8 and 10, respectively. Heat shock treatment decreased development of both Nelore and crossbred embryos. There was a significant interaction between time (12, 48 or 96 hpi) and temperature for blastocyst rates, i.e., the embryos became more thermotolerant as development proceeded. In Experiment 2, oocytes from Nelore and Holstein cows were fertilized with semen from bulls of either Nelore or Angus breeds, and subjected to 12 h HS at 96 hpi. Heat shock at 96 hpi, decreased embryo development. Additionally, cowxtreatment and bullxtreatment interactions were significant for blastocyst rates, i.e., both breed of cow and breed of bull affected the decline in blastocyst rate caused by heat shock treatment. In conclusion, the present results indicate that Nelore embryos (indicus) are more resistant to heat shock than Holstein (taurus) at early stages of in vitro development, and that embryos become more thermo-tolerant as development proceeds. Additionally, the resistance to heat shock was a result of the genetic contribution from both oocyte and spermatozoa.     

Ovarian function in Nelore (Bos taurus indicus) cows after post-ovulation hormonal treatments

Maternal recognition of pregnancy in the cow requires successful signaling by the conceptus to block luteolysis. Conceptus growth and function depend on an optimal uterine environment, regulated by luteal progesterone. The objective of this study was to test strategies to optimize luteal function, as well as prevent a dominant follicle from initiating luteolysis. Nelore (Bos taurus indicus) beef cows (n=40) were submitted to a GnRH/PGF(2alpha)/GnRH protocol. Cows that ovulated from a dominant ovarian follicle (ovulation=Day 0) were allocated to receive: no additional treatment (G(C); n=7); 3000IU of hCG on Day 5 (G(hCG); n=5); 5mg of estradiol-17beta on Day 12 (G(E2); n=6); or 3000IU of hCG on Day 5 and 5mg of estradiol-17beta on Day 12 (G(hCG/E2); n=5). Ultrasonographic imaging of the ovaries, assessment of plasma progesterone concentration, and detection of estrus were done daily from Day 5 to the day of subsequent ovulation. Treatment with hCG induced an accessory CL, increased CL volume, and plasma progesterone concentration throughout the luteal phase (P<0.01). Estradiol-17beta induced atresia and recruitment of a new wave of follicular growth; it eliminated a potentially estrogen-active, growing ovarian follicle within the critical period for maternal recognition of pregnancy, but it also hastened luteolysis (Days 16 or 17 vs. Days 18 or 19 in non-treated cows). In conclusion, the approaches tested enhanced luteal function (hCG) and altered ovarian follicular dynamics (estradiol-17beta), but were unable to extend the life-span of the CL in Nelore cows.     

Motility of the oviduct and uterus of the cow during the oestrous cycle.

Recordings of electrical activity of the oviduct and uterus were obtained during three oestrous cycles in cows fitted with an extra-cellular multi-electrode assembly. The stages of the cycle were identified by the appearance of the cervico-vaginal secretions and changes in the peripheral plasma level of progesterone were determined by radioimmunoassay. A gradual transition from local non-propagating electrical activity to propagating electrical activity with increase in the duration of contractions and then of their amplitude occurred 48 hr before the onset of oestrus. The transition coincided with a rapid decrease in progesterone level from 5 to 10 ng/ml to less than 0-1 to 0-4 ng/ml. This phenomenon was recorded from all uterine electrode sites, but was most marked at the uterotubal junction. Two days before oestrus, trains of potentials and bursts of activity became progressively grouped, apparently randomly, into prolonged phases in the distal portion of the oviduct and over the entire myometrium. During oestrus, the phases of activity became synchronized at these sites and both their amplitude and frequency reached a maximum. The strength but not the frequency of the phases diminished progressively 3 days after oestrus, followed by relative inactivity. The last remaining zone of activity was the uterotubal junction. During oestrus, the activities of the oviduct and the uterus were modified by oxytocin and adrenaline, the effect of the former being more marked on the uterus and that of the latter on the oviduct.     

Energy substrates in bovine oviduct and uterine fluid and blood plasma during the oestrous cycle

Up to 40 percent of cattle embryos die within 3 weeks of fertilization but there is little or no published information on the composition of the oviduct and uterine fluids essential for their survival during this time. We have measured the concentrations of the energy substrates, glucose, lactate, and pyruvate in cattle oviduct fluid on Days 0, 2, 4, and 6 and uterine fluid on Days 6, 8, and 14 of the oestrous cycle and corresponding blood samples. Oviduct and uterine fluids were collected in situ. Glucose concentrations in oviduct and uterine fluids were similar on all days and lower than in plasma (P < 0.05). Oviduct lactate concentration was up to eightfold higher than uterine or plasma concentration (P < 0.01). Oviduct pyruvate concentrations were similar on all days and lower than plasma concentrations on Days 0 and 2 (P < 0.005). Pyruvate concentrations were similar in the uterus and in plasma except on Day 14 when the concentration in plasma was higher (P < 0.05). There were no associations between systemic progesterone or oestradiol and glucose, lactate or pyruvate. There was a linear positive relationship (P < 0.001) between oviduct fluid secretion rate and oviduct glucose concentration and a linear negative relationship (P < 0.001) between oviduct fluid secretion rate and oviduct lactate, but no association between uterine fluid secretion rate and energy substrates. The different concentrations and associations between the energy substrates in oviduct and uterine fluids and blood plasma indicate a differential regulation of the secretion of these energy substrates by the oviduct and uterine epithelium.     

Tissue concentration, mRNA expression and stimulation of IGF-I in luteal tissue during the oestrous cycle and pregnancy of cows

The expression of IGF-I in bovine luteal tissue was demonstrated by parallel measurement of IGF-I tissue concentration and its mRNA; highest synthesis was observed during Days 12-17 of the cycle and the first months of pregnancy. Tissue levels of IGF-I increased from Days 1-5 to Days 12-17 of the cycle followed by a rapid decrease at luteolysis; there was a continuous decline from early pregnancy until Months 6-9. Microdialysis perfusion experiments with corpora lutea in vitro at Days 8-11 of the cycle revealed a major effect: release of progesterone and oxytocin were highly stimulated in a dose-dependent manner. We suggest that IGF-I could be important in regulating the function of the bovine corpus luteum and may act in an autocrine/paracrine way.     

Identical marker alleles in Podolic cattle (Bos taurus) and Indian zebu (Bos indicus)

In the context of biochemical marker research and in order to add new information on native breeds, the present work focuses on a local Southern Italy cattle, namely Italian Podolic. We provide the complete structural characterisation of alpha-lactalbumins and beta-globin chains isolated from Podolic cattle (Bos taurus). Given the unavailability of the complete sequence for alpha-lactalbumin A of taurine cattle in the literature, we intended to check its structure in order to ascertain the absence of any possible silent mutation. Screening the Podolic cattle, we found a new beta-globin variant not detectable by conventional methods. The presence of such a new variant might be helpful in the study of the Podolic population genetic structure and for a better knowledge of the gene pool per se, and in comparison with the other breeds. Structural analyses showed that the new beta-globin Podolic variant exhibited the same sequence as beta-globin Azebu. The alpha-lactalbumin A was the same as that isolated from zebu cattle (Bos indicus). The results are discussed in relation to the possible involvement of the two markers in the debate on the origin of the Podolic breed.     

Estimate of the population of preantral follicles in the ovaries of Bos taurus indicus and Bos taurus taurus cattle

The number of oocytes recovered from Bos taurus indicus females subjected to ovum pick-up averaged two to four times greater compared to Bos taurus taurus females. The objective of the present study was to test the hypothesis that this difference in oocyte yield was due to more preantral follicles in the ovaries of Bos indicus females. Ovaries (n = 64) from Nelore (Bos indicus) fetuses (n = 10), heifers (n = 12), and cows (n = 10), and Aberdeen Angus (Bos taurus) fetuses (n = 10), heifers (n = 12), and cows (n = 10) were cut longitudinally into halves, fixed, and processed for histological evaluation. The number of preantral follicles was estimated by counting them in each histological section, using the oocyte nucleus as a marker and employing a correction factor. The average number of preantral follicles in the ovaries of Bos indicus vs Bos taurus was (mean ± SD) 143,929 ± 64,028 vs 285,155 ± 325,195 for fetuses, 76,851 ± 78,605 vs 109,673 ± 86,078 for heifers, and 39,438 ± 31,017 vs 89,577 ± 86,315 for cows (P > 0.05). The number of preantral follicles varied greatly among individual animals within the same category, as well as between breeds. In conclusion, we inferred that the higher oocyte yield from Bos indicus females was not due to a greater ovarian reserve of preantral follicles. Therefore, mechanisms controlling follicle development after the preantral stage likely accounted for differences between Bos indicus and Bos taurus females in number of oocytes retrieved at ovum pick-up.     

Electrophoretic characterization of proteins in oviduct fluid of cows during the estrous cycle

This study was undertaken to examine the in vivo protein composition of the bovine oviduct during the estrous cycle. Oviduct fluid was collected daily from four oviduct-cannulated dairy cows for a total of four complete estrous cycles. Fluid secretion followed a definite cyclic pattern, with maximum secretion occurring at estrus in all cycles. Protein concentration fluctuated during the cycle and varied among animals. In general, protein concentration was lower at the time of estrus. Total protein in oviduct fluid, however, was higher around estrus, indicating increased transudation or secretion by the oviduct. One-dimensional SDS-PAGE separation revealed the protein pattern of oviduct fluid to be generally similar to that of blood serum and follicular fluid. Two proteins appeared to be oviduct-specific. The first, a protein of approximately 47 kDa, was evident in oviduct fluid throughout the estrous cycle. The second protein, evident as a broad diffuse staining band above albumin, appeared for only 3-4 days at or near ovulation. This protein had a molecular weight of 80-95 kDa and stained positive for carbohydrate with periodic acid-Schiff reagent. These studies indicate that the in vivo protein composition of oviduct fluid varies with the estrous cycle, and that around estrus, an oviduct-specific glycoprotein is present.     

High environmental temperature and humidity decrease oocyte quality in Bos taurus but not in Bos indicus cows

Two experiments were conducted to assess the effects of environmental temperature and humidity on the quality and developmental capabilities of bovine oocytes. In Experiment 1, Bos taurus (Holstein and crossbred Angus) cows were subjected to 5 weekly sessions of ultrasound-guided follicle aspiration from February 16 through March 23 (cool season) and 5 sessions from May 22 through June 20 (hot season). In Experiment 2, Bos taurus (Holstein) and Bos indicus (Brahman) cows were superstimulated (Super-Ov) during the months of August (hot season) or January (cool season), and each cow was subjected to a single oocyte aspiration session. In each experiment, oocytes were classified as normal or abnormal based on ooplasm morphology and cumulus cell layers. In Experiment 1, oocytes classified as normal were in vitro matured and fertilized (IVM/IVF), and the resulting embryos cultured for 8 d. All oocytes recovered from superstimulated cows in Experiment 2 were matured and fertilized in vitro and the subsequent embryos cultured for 8 d, regardless of their morphological appearance. In Experiment 1, Bos taurus cows produced a higher (P = 0.02) percentage of normal oocytes during the cool season (75.9 +/- 8.0) than during the hot season (41.0 +/- 9.5). The percentage of fertilized oocytes developing to the 2-cell (82.4), 8-cell (65.4) and morula (46.6) stages were also greater (P < or = 0.06) during the cool season than the hot season (45.0, 21.2, 6.0 for 2-cell, 8-cell and morula stages, respectively). In Experiment 2, Bos taurus cows (Holstein) had a lower (P = 0.01) percentage of normal oocytes in the hot season (24.5 vs 80.0) and a lower (P < or = 0.003) percentage of fertilized oocytes developing to the 8-cell, morula and blastocyst stages. No difference (P > or = 0.57) in the percentage of normal oocytes or in embryo development was detected between seasons in Bos indicus (Brahman) cows. In conclusion, high environmental temperature and humidity resulted in a marked decline in the quality of oocytes retrieved from Bos taurus cows and markedly decreased their in vitro developmental capabilities. In contrast, a high percentage of oocytes retrieved from Bos indicus cows exhibited normal morphology and yielded a high proportion of blastocysts, regardless of season.     

Comparative analysis of GDF 8 (myostatin) in Bos indicus and Bos taurus.

We report the myostatin gene sequence of Bos indicus cattle in comparison to Bos taurus. B. indicus genomic sequence was obtained by overlapping PCR amplification of genomic DNA. Exon splice sites were confirmed by mRNA sequencing. There were 5 exonic single nucleotide polymorphisms (SNP) only one of which was a non-synonymous mutation that resulted in a serine to asparagine (S214N) amino acid substitution. The B. indicus gene has two insertions of 16 and 12 bases in the first intron. In addition, SNPs in the 3' UTR and intronic regions are also reported.     

Immunophenotype of porcine oviduct epithelial cells during the oestrous cycle: a double-labelling immunohistochemical study

The luminal epithelium of the porcine oviduct is composed of ciliated cells and secretory cells, but it is assumed for several species that under the control of steroid hormones secretory cells are able to be transformed into ciliated cells. In order to better understand such physiological changes during the different stages of the oestrous cycle, we evaluated epithelial cell proliferation together with oestrogen receptor (ER) expression of porcine ampullary oviducts. To identify the immunophenotype of proliferating cells, double immunohistochemistry was performed using anti-chromogranin A antibody (anti-CgA) as the second primary antibody. Anti-CgA, recently shown to be an immunocytochemical marker of ciliated cells of the cow, also labelled specifically the luminal surface of ciliated cells of the pig. Double labelling of sections with the monoclonal antibody MIB-1 against the proliferation-associated nuclear epitope Ki-67 and anti-CgA clearly demonstrates that MIB-1 was selectively localised in the nuclei of secretory cells. Proliferative activity was not observed in CgA-positive ciliated cells in all examined stages of the oestrous cycle. The percentage of Ki-67-positive epithelial cells was higher at pro-oestrus, compared with the other stages of the oestrous cycle. Furthermore, ER immunoreactivity was exclusively detected in the nuclei of the epithelial cells, which were negative for CgA. We conclude, therefore, that oestrogen may induce the initial proliferation of secretory cells and promote the differentiation into ciliated cells.     

Follicular dynamics during postpartum anestrus and the first estrous cycle in suckled or non-suckled Brahman (Bos indicus) cows

Brahman (Bos indicus) cows, were selected at 28+/-10 days after calving and analyzed by real time rectal ultrasonography three times a week, in order to evaluate and compare follicular and corpus luteum development during postpartum (PP) anestrus and the first PP estrous cycle under sylvopastoril conditions. Suckling (S, n=11) or non-suckling (NS, n=5) cows were evaluated in a zone of tropical dry forest (450m of altitude, mean temperature=27 degrees C, annual rainfall=1000mm). Estrous detection was performed twice daily by direct observation. Progesterone was quantified using RIA. From 28+/-10 days postcalving to resumption of estrous cycles, there were no differences (P>0.05) between NS and S cows for diameter of the dominant or first subordinate follicle, follicular growth rate, or interdominance interval. Silent ovulation, corpus luteum formation and subsequent progesterone concentrations ranging from 0.3 to 9. 7ng/ml, were found in both groups. The first calving to ovulation and calving to standing estrus intervals were shorter (P<0.01) in NS (34.8+/-5.81 and 41.2+/-9.03 days) than in S (65+/-4.82 and 81+/-6. 21 days) cows. Follicular development and progesterone concentrations during the first PP estrous cycle did not differ (P>0. 05) between NS and S cows. These results suggest that Brahman cows could have an early PP resumption of follicular recruitment if fed under sylvopastoril system conditions. However, non-suckled cows did have an earlier standing estrus and ovulation than did suckled cows.     

Fertility on synchronized estrus in crossbred (Bos taurus x Bos indicus) heifers

This study was performed in Western Maharastra (India). The 272 crossbred heifers were randomly allocated within herds to one of the four following groups: 1) silastic coils, 10 days treatment (n = 65); 2) Norgestomet implants, 10 days treatment (n = 71); 3) prostaglandin F(2alpha) two injections 11 days apart (n = 70); and 4) control (n = 66). Almost all heifers in the treated groups were detected in heat during the four days following treatment (88-100%) vs 26% in the control group in the first 21 days. Mean conception rates at first AIs were respectively 62,48 and 60% in groups 1,2 and 4 (p > 0.05) and only 29% in group 3. By 90 days after treatment, 66, 59, 46 and 33 per cent of the females were pregnant for groups 1 to 4, respectively (p < 0.001). In conclusion, progestogen treatments seemed to be highly satisfactory both in terms of conception rates and intervals from treatment to pregnancy.     

云南黄牛和大额牛的mtDNA多态性研究

本文以mtDNA限制性内切酶片段长度多态技术分析云南牛的mtDNA多态性。结果表明云南黄牛有两种类型的mtDNA分子,一种是普通黄牛类型,另一种是瘤牛类型,两者的频率分别为33%和67%。没有发现过渡型和重组型。昆明黑白花奶牛的mtDNA与云南黄牛的相类似。云南黄牛可能具有两种起源,即普通黄牛起源和瘤牛起源。今天的云南黄牛群体是这两种类型的混合。大额牛的限制性类型与瘤牛相同,表明大额牛的起源与瘤牛有密切关系。