Seasonal Profiles of Ovarian Activity in Iberian lynx (Lynx pardinus) Based on Urinary Hormone Metabolite Analyses

The Iberian Lynx Ex-Situ Conservation Programme is an essential part of a co-ordinated action plan to conserve the most endangered felid species of the world. Successful captive breeding demands reliable methods for reproduction monitoring including reliable non-invasive pregnancy diagnosis. During a 3-year study, urine samples from six captive Iberian lynx females were obtained (one non-pregnant, one pseudo-pregnant and 11 pregnant cycles). Progesterone, pregnanediol and oestradiol were determined in urinary extracts and relevant urinary oestrogen metabolites were characterized by high-performance liquid chromatography (HPLC). Urinary progestins did not follow the typical pregnancy-related course of felids. In the lynx, we failed to demonstrate an urinary progestin elevation during pregnancy. In contrast, urinary oestrogens increased from 3.8 ± 0.6 to 8.6 ± 0.5 ng/mg creatinine (p < 0.001) during the pregnancy. A comparison of pseudo-pregnant with pregnant cycles revealed a further increase of oestrogens caused by implantation (p < 0.05). In one female, which refused to mate, no difference was estimated between oestrogens levels during the breeding and non-breeding seasons. Almost 10-fold higher oestrogen concentrations were measured in urines of females that shared enclosures with males. HPLC analysis of oestrogens in urine samples collected from Iberian lynx during the pregnancy revealed that lynx urine is composed of two polar oestrogen metabolites in addition to oestrone and minor amounts of oestradiol. Oestrone was detectable in all urinary extracts (8–12% of metabolites), whereas oestradiol was elevated only during late pregnancy (18%). Thus, seasonal luteal activity in Iberian lynx can be monitored by urinary oestrogens. The increase of urinary oestradiol during late pregnancy might indicate an oestradiol secretion by the lynx placenta.     

An Experimental Model to Study Resistance Index and Systolic/Diastolic Ratio of Uterine Arteries in Adverse Canine Pregnancy Outcome

The aim of this study was to describe the changes in the resistance index (RI) and systolic/diastolic ratio ( S / D ) of the uterine arteries during mid-pregnancy abortion induction in the dog. Sixteen 30–35 day pregnant bitches were randomly assigned to either a pharmacological protocol to interrupt gestation (n = 8) or were used as untreated control group (n = 8). Doppler assessments of uterine arteries blood flow were carried out before the initiation of the protocol and then every other day up to abortion (treated group) or parturition (control group). All treated bitches aborted 6 ± 1.2 days after initiation of the treatment (while none of the non-treated bitches aborted). Pre-treatment RI and S / D did not differ between groups (p > 0.2) while average post-treatment indexes were (mean ± SD): 0.62 ± 0.1 vs 0.53 ± 0.1 (p < 0.01) and 2.96 ± 0.9 vs 2.23 ± 0.3 (p = 0.01), for the treated and non-treated group respectively. Correlations between days to abortion and RI or S / D were 0.75 (p < 0.01) and 0.79 (p < 0.01) and, −0.78 (p < 0.01) and −0.73 (p < 0.01) for the treated and non-treated groups respectively. In the treated group, correlations between serum progesterone (P₄) concentrations and RI and S / D were −0.76 (p < 0.01) and −0.59 (p < 0.01) respectively. It is concluded that, during induction of abortion, RI and S / D of uterine arteries progressively increased while P₄ decreased.     

Influence of gestation on blood plasma concentrations of oestrone and oestrone sulphate in Karan Swiss cows and Murrah buffaloes

Single blood samples from 106 pregnant and seven non-pregnant Karan Swiss cows and 104 pregnant and nine non-pregnant Murrah buffaloes were measured for oestrone and oestrone sulphate hormones by radioimmunoassay. Mean plasma oestrone level was below detection limit (less than 2.5 pg/ml) in non-pregnant and 1 month pregnant cows and buffaloes. In cows the mean oestrone level fluctuated narrowly between 10.25 and 26.65 pg/ml between the second and eight months of pregnancy, followed by a steep rise in the ninth and especially in the tenth month (151.24 pg/ml). In buffaloes mean oestrone concentrations were lower and fluctuated between 14.81 and 23.56 pg/ml during the second to ninth months of pregnancy, rising sharply in the tenth month to a peak of 47.37 pg/ml. Mean oestrone sulphate levels were below detection limit (less than 16 pg/ml) during non-pregnancy, first and second months of pregnancy in cows, increasing sharply thereafter to a peak of 6401.38 pg/ml in the tenth month of pregnancy. In buffaloes, low mean levels of oestrone sulphate were recorded in the non-pregnant and up to the fourth month of pregnancy with the levels rising sharply thereafter to a peak of 6559.82 pg/ml in the tenth month. The hormone levels were not significantly different in the two species (P greater than 0.01). The possibility of using oestrone sulphate measurement as a test of pregnancy confirmation has been indicated for both species.     

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

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.     

Comparison of Two Doses of the GnRH Antagonist, Acyline, for Pregnancy Termination in Bitches

Gonadotropin-releasing hormone (GnRH) antagonists are particularly useful when a rapid inhibitory effect on the gonadal axis is required. The aim of this study was to test the efficacy and clinical safety of a low and high dose of the third generation GnRH antagonist, acyline, on pregnancy termination in female dogs. The effect of the antagonist on the progesterone (P₄) serum concentration was also described. Twenty-one mid-pregnant bitches were randomly assigned to a single subcutaneous (SC) dose of a placebo (PLACE; n = 7), a low (ACY-L; 110 μg/kg; n = 6) or high (ACY-H; 330 μg/kg; n = 8) dose of acyline. The animals were followed up for 15 days. All ACY treated but no placebo-treated animals terminated their pregnancy by abortion (p < 0.01). The ACY-L and ACY-H groups interrupted their pregnancy 7 ± 1.9 and 6.4 ± 1.3   days after treatment, respectively (p = 0.7). A significant interaction between treatment and day was found (p < 0.01) for P₄ serum concentrations when PLACE was compared with both ACY groups. No difference was found for this hormone between both ACY groups (p > 0.05) where P₄ diminished throughout the study. The decreasing rate varied among animals and was closely related to the time of abortion when P₄ reached basal concentrations. In PLACE animals, gestation progressed normally and P₄ did not change throughout the study (p > 0.05). None of the bitches presented side effects. It was concluded that acyline safely terminated mid-pregnancy by permanently decreasing P₄ serum concentrations.     

Accuracy of transrectal palpation for early pregnancy diagnosis in Egyptian buffaloes

The present study was undertaken to evaluate the accuracy of transrectal palpation (TRP) for diagnosing early pregnancy in buffaloes and the false diagnoses of the TRP test by using the pregnancy-associated glycoprotein radioimmunoassay (PAG-RIA) test. Pregnancy was diagnosed in 168 buffalo–cows once by TRP and PAG-RIA test between days 31 and 55 after breeding. The sensitivity of TRP for detecting pregnant buffalo–cows was 37.5% at days 31–35, increased to 93.8% at days 46–50 and reached 100% at days 51–55 (P < 0.01). All cases of false negative diagnoses (n = 10) had PAG concentration higher than the threshold (≥1.8 ng/mL) for diagnosing pregnancy. The specificity of TRP for detecting non-pregnant buffalo cows ranged between 90.9%, and 100% between days 31 and 55. All cases of false positive diagnoses (n = 5) made by TRP had PAG concentrations lower than the threshold for diagnosing pregnancy. It could be concluded that TRP is an accurate method for diagnosing pregnant and non-pregnant buffalo cows from day 46 after breeding.     

Single and multiple-dose pharmacokinetics of tepoxalin and its active metabolite after oral administration to rabbits (Oryctolagus cuniculus)

The anti-inflammatory agent, tepoxalin, was administered to eight healthy 6-month-old female New Zealand white rabbits once daily at an oral dose of 10 mg/kg. Blood samples were obtained immediately before and at 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 h postadministration on days 1 and 10. Tepoxalin and its active metabolite, RWJ 20142, concentrations were determined in plasma by use of high-performance liquid chromatography with mass spectrometry. C _max of the parent compound was reached between 3 and 8 h of drug administration, with a harmonic mean t _1/2 of 3.6 h. Peak tepoxalin plasma concentrations were 207 ± 49 ng/mL. After oral administration, the metabolite RWJ 20142 achieved C _max in plasma 2–8 h after administration, with a t _1/2 of 1.9–4.8 h (harmonic mean 2.8 h). Peak plasma concentrations of RWJ 20142 on day 1 were 2551 ± 1034 ng/mL.     

Establishment of an ELISA for Measuring Bovine Pregnancy-Associated Glycoprotein in Serum or Milk and Its Application for Early Pregnancy Detection

It has been shown in several mammalian species that during pregnancy, trophoblast cells express a range of pregnancy-associated glycoproteins (PAG). The presence of PAG in the maternal serum of cows may serve as an indicator of pregnancy from day 28 after AI onward. The present study addresses (1) conversion of an existing PAG-RIA to a competitive double antibody ELISA using a polyclonal anti-bPAG-IgG and an anti-rabbit-IgG raised in sheep for coating and (2) application of newly established ELISA to test its suitability for pregnancy detection by measuring PAG in serum or milk. The intraassay coefficients of variation (CV) for the PAG-ELISA were 2–14% for serum and 10–12% for milk; the corresponding interassay CVs were 8–22% and 12–22%, respectively. Pregnancy-associated glycoprotein profiles established in milk and serum of 12 pregnant cows showed a characteristic pattern with measurable amounts from approximately day 20 onwards in serum and from day 60 onwards in milk. In a field trial, serum PAG was determined in 397 cows sampled between 20 and 50 days after insemination. The outcome was that, pregnancy could reliably be diagnosed from day 28 onwards in serum and from day 150 onwards in milk. In conclusion, it may be stated that the established ELISA provides an efficient and reliable means of pregnancy diagnosis that will, in our judgement, gain in popularity with cattle breeding. The ELISA proved to be an adequate and efficient way of measuring PAG in maternal serum or milk and will be a useful means of pregnancy detection in cows.     

Progesterone and pregnancy status of buffaloes treated with a GNRH agonist

The primary aim of the present study was to establish whether the treatment with a GnRH agonist on Day 5 after AI may result in the formation of an accessory corpus luteum, greater progesterone secretion, and the increased likelihood of pregnancy success in buffaloes. The study was conducted during a period of increasing daylight length when progesterone secretion is suppressed and embryonic mortality is relatively high in buffaloes. In Experiment 1, treatment with a GnRH agonist (buserelin, 12 μg) on Day 5 after AI induced acute increases in circulating concentrations of LH, FSH and oestradiol-17β. Pregnant buffaloes (n = 14) at Day 40 following AI showed an increase (P < 0.01) in milk whey progesterone concentration between Day 5 (310 ± 55 pg/ml) and Day 15 (424 ± 50 pg/ml). The non-pregnant buffaloes (n = 7) showed a decrease (P < 0.01) in progesterone level from Day 5 (410 ± 87 pg/ml) to Day 15 (188 ± 30 pg/ml) following AI. In Experiment 2, the treatment with buserelin (12 μg) on Day 5 after AI induced ovulation in 62% of the buffaloes (31/50) and these buffaloes showed a progressive increase in milk whey progesterone concentration on Day 10, 15 and 20 of pregnancy. Buffaloes that did not ovulate, recorded a relatively constant milk whey progesterone level from Day 10 to Day 20 following AI. Milk whey progesterone concentrations increased after the administration of the GnRH agonist in 97% of the pregnant buffaloes and 68% of the non-pregnant buffaloes. The diameter of the largest follicle in buffaloes that ovulated (ovulated n = 31) (8.9 ± 0.04 mm; range 4.2 – 13.0 mm) did not differ significantly from the diameter of the largest follicle in buffaloes that did not ovulate (not ovulate n = 19) (8.7 ± 0.04 mm; range: 4.0 – 12.0 mm). The latter observation suggested that notional ovulatory size follicles in buffaloes are heterogeneous with respect to stage of follicle maturation and capacity to respond to plasma LH. The present study showed that treatment with a GnRH agonist on Day 5 following AI provides a strategy to increase progesterone secretion and the likelihood of pregnancy in buffaloes mated during periods of increasing daylight length.     

Enzymeimmunoassay of oestrone sulphate concentrations in faeces for non-invasive pregnancy determination in mares

AIM: To determine the suitability of measuring faecal oestrone sulphate (OS) by enzymeimmunoassay as a means of determining pregnancy status in mares bred under New Zealand conditions. METHODS: An antibody-coated microtitre plate-based enzymeimmunoassay was used to determine the concentration of OS in faecal and plasma samples obtained from pregnant and non-pregnant mares. RESULTS: In non-pregnant mares, the mean faecal OS concentration was 34 ng/g, and the value three standard deviations above this was 80 ng/g. None of 427 faecal samples collected from 116 non-pregnant mares over a l-year period had an OS concentration >80 ng/g. Only five samples from three mares had an OS concentration >65 ng/g, the value two standard deviations above the mean non-pregnant value. Analysis of faecal OS concentrations in 532 faecal samples collected from 39 pregnant mares showed that as pregnancy progressed, an increasing proportion of faecal samples had OS concentrations >80 ng/g. None of the mares 150 days or more pregnant had faecal OS concentrations <50 ng/g, and 204/220 samples obtained from these mares had faecal OS concentrations >80 ng/g. Following foaling or foetal death, elevated faecal OS concentrations returned quickly to non-pregnant levels. The mean +/- s.e.m. plasma level of OS in five mares bled daily throughout one oestrous cycle was 1.7 +/- 0.2 ng/ml. Sixty-eight blood samples from pregnant mares bled up to five times between 92 days after mating and foaling all had plasma OS concentrations >30 ng/ml, with 64/68 being >50 ng/ml. CONCLUSIONS: This study shows that measuring faecal OS concentrations by enzymeimmunoassay offers a convenient, accurate, non-invasive means of determining pregnancy status in mares from 150 days after mating onwards. Mares with faecal OS concentrations <50 ng/g can be considered not pregnant, while mares with faecal OS concentrations >80 ng/g can be considered pregnant. Those few mares returning a faecal OS concentration between 50 and 80 ng/g should be retested to obtain a conclusive result. Measuring plasma OS concentrations allows pregnancy status to be determined earlier (from 100 days after mating). Moreover, the discrimination between non-pregnant and pregnant levels is greater for OS in plasma than in faeces. CLINICAL RELEVANCE: Measurement of OS concentrations in faeces provides an alternative and non-invasive means of determining pregnancy status in mares from 150 days after mating.     

OC4Influence of Seminal Plasma Added to Highly Diluted Semen on Sperm Motility of Young Boar Feed with Selenium and Vitamin E

High dilution rates have been documented as detrimental for boar spermatozoa, shortening their lifespan (Centurion et al. 2003, Biol Reprod 69: 640–646). Addition of seminal plasma (SP) to semen extenders, or selenium (Se) and vitamin E (VE) in diet of boars could increase motility of highly diluted spermatozoa (HDS). The aim of this work was to evaluate the effect of seminal plasma on sperm motility of HDS from boars feed with Se and VE. Sixteen 12 month-old boars were designed to one of four dietary treatments: (i) control, Se 0 ppm–VE 0 IU/kg; (ii) Se 0–VE 250; (iii) Se 0.5–VE 250 and (iv) Se 0.5–VE 0. Boars were treated for 8 weeks before semen collection. Sperm rich fractions from each boar were diluted to 5 × 10⁶ sperm/ml in PBS medium and incubated at 37°C with or without 10% SP. The measurements were done at 0, 2 or 5 h. Data were analyzed as a mixed model for a factorial design [2 (Se) × 2 (VE) × 2 (SP) × 3 (h)]. Percentage of sperm motility (PSM) increases significantly (p < 0.001) with addition of Se (81.3 ± 1.52), VE (81.0 ± 1.62) and SP (81.5 ± 1.57) vs control (73.4 ± 1.61). There was significant interaction Se × VE (p < 0.001) and Se × VE × SP (p < 0.05) in PSM. However, PSM was affected significantly by time (0 h 83.4 ± 1.92; 2 h 80.7 ± 1.92 and 5 h 67.9 ± 1.92; p < 0.001). There was significant interaction SP × Time (p < 0.05) in PSM. These results indicate that Se, VE and SP improve seminal viability. Addition of 10% of SP maintains PSM at least during 5 hours.     

Plasma Progesterone Profile in Ovariectomized Beef Cows after Intra-vaginal Insertion of New, Once-used or Twice-used CIDR

Objective of this study was to show plasma progesterone concentrations in ovariectomized beef cows after treatment with new, once-used and twice-used controlled internal drug-releasing devices (CIDRs). Four ovariectomized beef cows were used for the experiment. Plasma concentrations of progesterone were quantified using a validated ELISA. The CIDR was inserted into vagina of cows by using a standard CIDR applicator and then removed 7 days after insertion. One week later, once-used CIDR was inserted and removed on day 7. Twice-used CIDR was, then inserted at an interval of 7 days. Mean plasma concentrations of progesterone 24 h after new CIDR insertion was 4.0 ± 0.1 ng/ml, which thereafter decreased gradually to 1.4 ± 0.1 ng/ml at day 7. In cows treated with once-used CIDR or twice-used CIDR, mean plasma progesterone concentrations at day 1 were 2.4 ± 0.2 or 1.8 ± 0.2 ng/ml and 1.0 ± 0 or 0.9 ± 0.1 ng/ml at day 7 respectively. The results suggest that once-used CIDR may be still effective to produce luteal phase progesterone concentrations in plasma in non-suckling beef cows.     

Riverine status and genetic structure of Chilika buffalo of eastern India as inferred from cytogenetic and molecular marker-based analysis

The present study aimed at assessing the status of the Chilika buffalo population of eastern India employing cytogenetic and molecular markers. The Chilika buffaloes investigated cytogenetically possess a somatic chromosome count of 50, identical to that of typical riverine buffaloes. Various diversity estimates, viz. observed number of alleles (4.68), effective number of alleles (2.79), and observed (0.487) and expected (0.602) heterozygosity across 25 heterologous microsatellite markers indicated the presence of a moderate level of genetic diversity in Chilika buffaloes, comparable with three other prominent Indian riverine buffalo breeds (Murrah, Nagpuri and Toda) included in this study. Across the four buffalo populations, mean estimates of F -statistics from Jackknifing over loci were significantly different from zero (p < 0.05), with F _IT (total inbreeding estimate) = 0.315 ± 0.038, F _IS (within-population inbreeding estimate) = 0.178 ± 0.038, and F _ST (population differentiation) = 0.166 ± 0.025. Inter-breed analysis reflected Chilika buffaloes to be genetically close to Nagpuri followed by Murrah and Toda buffaloes. Factorial correspondence analysis (FCA) revealed low breed-specific clustering of Chilika and Nagpuri buffaloes. Additionally, the neighbour-joining tree structure of mitochondrial DNA D-loop haplotypes indicated clear grouping of the Chilika haplotypes with the riverine buffalo. Thus the cytogenetic, microsatellite and mitochondrial data analysed in the present study classify Chilika buffalo of eastern India to be of the riverine type and not swamp-type buffalo.     

Isolation and Culture of Ovine and Bubaline Small and Large Pre-antral Follicles: Effect of Cyclicity and Presence of a Dominant Follicle

Studies were conducted to examine the effects of the cyclicity and the presence of a dominant follicle (DF) in ovary on the recovery and in vitro growth of pre-antral follicles (PFs) in sheep and buffalo. Small pre-antral follicles (SPFs, 100–250 μm) and large pre-antral follicles (LPFs, 250–450 μm) were isolated from slaughterhouse ovaries in the breeding seasons by a mechanical and enzymatic method. The sheep and buffalo PFs were cultured in vitro for 6 and 15 days, respectively, and examined for their growth, survival and antrum formation rates and growth rates of oocytes in cultured pre-antral follicles. The follicles of the sheep and buffalo were recovered and cultured simultaneously within replicates. The recovery rates (number per ovary) of both SPFs and LPFs were significantly (p < 0.05) higher in cyclic ewes (SPFs: 22.0 ± 3.3 vs 12.1 ± 2.6 and LPFs: 16.0 ± 3.6 vs 9.2 ± 1.8) and buffaloes (SPFs: 9.2 ± 1.3 vs 4.1 ± 1.0 and LPFs: 10.3 ± 2.7 vs 5.4 ± 0.7) compared with those recovered from acyclic ones. Presence of a DF in ovary significantly (p < 0.05) reduced the recovery rates of LPFs in ewes (9.06 ± 2.7 vs 16.4 ± 3.8) but had no effect in buffalo. Cyclicity of animals or follicular dominance had no effects on in vitro growth, survival and antrum formation rates and growth rates of oocytes in cultured PFs of SPFs and LPFs in both sheep and buffalo. The in vitro growth, survival and antrum formation rates of LPFs and growth rates of oocytes in cultured LPFs were significantly (p < 0.05) higher than those observed in SPFs in both sheep and buffalo. The overall recovery and growth rates of the PFs were lower in buffaloes compared with ewes.     

Effects of Exogenous Tumour Necrosis Factor-α on the Secretory Function of the Bovine Reproductive Tract Depend on Tumour Necrosis Factor-α Concentrations

The aim of study was to correlate tumour necrosis factor-α (TNF) infused doses used with the TNF concentrations achieved and with the secretory function of both the ovary and the uterus in cows. We evaluated the concentrations of progesterone (P4), prostaglandin (PG)F_2α, PGE₂ nitric oxide (NO) and TNF in the jugular vein and vena cava caudalis as parameters of exogenous TNF action on the female reproductive tract. Aortae abdominalis of cows (n = 18) were infused with saline or two doses of TNF (luteolytic – 1 μg or luteotrophic – 10 μg). In the peripheral blood, 1 μg TNF concentrations achieved within the range of 30–45 pg/ml, and 10 μg TNF provoked a sharp increase in achieved concentrations at a range of 250–450 pg/mL). The TNF concentrations achieved in vena cava caudalis were five to six times higher than that in peripheral blood (p < 0.001). One microgram TNF increased PGF_2α and NO (p < 0.001) and decreased P4 (p < 0.05). The higher TNF dose stimulated P4 and PGE₂ (p < 0.01). TNF infusion at luteolytic dose achieved its concentrations at the physiological range previously observed in cows. Luteotrophic TNF dose achieved the concentrations in vena cava caudalis that are much higher than physiological level and were previously noted in pathological circumstances (i.e. mastitis , metritis ).     

Reproduction in the European Mink, Mustela lutreola: Oestrous Cyclicity and Early Pregnancy

Despite efforts undertaken to conserve the endangered European mink, its reproduction is still poorly studied. The aim was to study its reproductive cyclicity, faecal progesterone concentration and ovarian changes during early pregnancy, with the emphasis on the pre-implantation period and implantation. During the 2004 breeding season, oestrous cycle was monitored in 39 females as well as ovarian changes during early pregnancy in 22 females. During the 2007 breeding season, faecal progesterone concentration measured by radioimmunoassay was monitored during pregnancy in 10 females throughout their pregnancy. The breeding season 2004 started on March 18 and ended on May 10, with the peak recorded in April. The duration of first oestrus was 1–12 days. If not mated, the vast majority of females entered second oestrus after 12–55 days. In general, relatively low faecal progesterone values were detected in European mink; an average of 42.69 ± 4.70 ng/g faeces in oestrous females with a maximum of 176.44 ± 23.01 ng/g faeces on pregnancy day 12. anova indicated a significant effect of the pregnancy stage. Post hoc comparisons with Fisher least significant difference (LSD) test revealed that faecal progesterone concentrations on days 8 and 12 post coitum (p.c.), but not at the end of pregnancy (day 40), were higher when compared with the initial oestrous level. Implantation in this species occurs on day 12 p.c. and was indicated by prominent uterine swellings and failure to flush the uterine horns beyond this day. Advanced luteogenesis was observed with prominent corpora lutea found in ovaries around the time of implantation. To conclude, European mink is a seasonally polyoestrous species; the early pregnancy of European mink resembles that of European polecat, i.e. in both species, implantation occurs on day 12 p.c. without any implantation delay.     

Measurement of Ovine Pregnancy-Associated Glycoprotein (PAG) During Early Pregnancy in Lacaune Sheep

This study describes ovine pregnancy‐associated glycoprotein (ovPAG) concentrations in 20 Lacaune sheep during early pregnancy. Measurements were performed by using semi‐purified ovPAG as standard, tracer and immunogens for antibody production in rabbits. Antisera R780 (against ovPAG_57+59kDa) and R805 (against ovPAG5_58+61kDa) were used respectively in RIA‐780 and RIA‐805. Blood samples were collected at days 0, 18, 20, 22 and 25 after artificial insemination. From day 18 after breeding onward, the mean ovPAG concentration was significantly higher (p < 0.001) in plasma samples from pregnant ewes (n = 17) than in non‐pregnant ones (n = 3). The specific activity of the tracer was 11 760 Ci/mmol in RIA‐780 and 14 900 Ci/mmol in RIA‐805. The minimal detection limits for RIA‐780 and RIA‐805 were 0.2 ng/ml and 0.3 ng/ml, respectively. The intra‐assay CV of samples with low (1.0 ng/ml), medium (2.5 ng/ml) and high (4.0 ng/ml) PAG concentrations were 3%, 6% and 9% for RIA‐780 and 8%, 9% and 5% for RIA‐805. The inter‐assay CV in the same samples were 13%, 12% and 7% for RIA‐780 and 13%, 11% and 5% for RIA‐805. The recovery was higher than 95% in both assays. No cross‐reaction was observed with members of aspartic proteinase family as well as with other tested proteins. In both RIA‐780 and RIA‐805, inhibition of the binding of the tracer by antisera was parallel between standard curve and serial dilutions of pregnant ewe samples. In conclusion, the two homologous RIA systems are suitable for early quantification of ovPAG concentrations in ewe plasma samples from day 18 after breeding.     

Changes in Blood Glucose,Plasma Non-esterified Fatty Acids and Insulin in Pregnant and Non-pregnant Goats

The blood glucose and the plasma non-esterified fatty acids (NEFA) and insulin concentrations were estimated in jugular blood samples from 18 Alpine×Beetal and Sannen×Beetal goats during pregnancy and compared with samples from non-pregnant goats and from goats during the periparturient period. The blood glucose levels in the pregnant goats rose to a peak of about 60±1.36 mg/ml at 42–56 days and then declined to about 46±2.37 mg/ml at 112–126 days. In non-pregnant goats, the blood glucose levels were significantly (p<0.01) higher than in pregnant goats, except between days 42 and 70 (59±1.36 mg/ml). On the day of kidding, the levels declined significantly (p<0.01), increasing again thereafter. The plasma NEFA concentrations were significantly higher in pregnant than in non-pregnant goats from days 56 to 126. The NEFA concentration increased on the day of kidding, followed by a transient fall by day 3. The plasma insulin concentration was usually higher in pregnant than in non-pregnant goats, except between days 56 and 70 and from day 126 onwards. The insulin concentration fell late in pregnancy, but there was a transient increase 2 days after parturition. The blood glucose and plasma NEFA concentrations can be used as indices of nutritional status during pregnancy in goats.     

Early Breeding and Pregnancy Diagnosis in Syrian Awassi Sheep Yearlings

Contents
Fifty-nine female yearlings of local Awassi sheep were randomly divided into two groups. Animals in group T (treated) were fitted with intravaginal sponges containing 60 mg of medroxyprogesterone acetate for 14 days followed by 400 IU of pregnant mare's serum gonadotrophin (PMSG) at sponge withdrawal, whereas group C (control) received no treatment. Oestrus rate was 92.7 and 11.2% for groups T and C, respectively. Lambing rate was 78 and 5.6% for groups T and C, respectively. Twinning rate was 31.3% in group T compared to zero in group C. Average birth weight for single born lambs (4.7 ± 0.6 kg) was significantly (p < 0.05) higher than twin born lambs (3.0 ± 0.5 kg) in group T. The average concentration of blood progesterone collected between days 17–19 after mating was 19.30 nmol/l and the accuracy of early pregnancy diagnosis was 100%. It was concluded that, it is possible to induce synchronized oestrus, and to increase the twinning rate in Syrian Awassi sheep yearlings outside the breeding season, using intravaginal sponges and PMSG. In addition, early pregnancy diagnosis could be successfully determined in female Awassi sheep yearlings between days 17–19 after mating.     

Expression of MHC-I and -II in Uterine Tissue from Early Pregnant Bitches

The aim of the study was to investigate the expression of major histocompatibility complex (MHC)-I and -II in uterine tissues from pregnant and non-pregnant bitches, taken at different time periods after mating. The pregnant bitches were ovariohysterectomized during the pre-implantation (group 1, n = 4), implantation (group 2, n = 7) and placentation stage (group 3, n = 7). Non-pregnant animals in diestrus served as controls (group 4, n = 7). The expression of MHC- I and -II in salpinx, apex, middle horn, corpus uteri and at implantation sites was investigated by immunohistochemistry as well as qualitative and quantitative RT-PCR; MHC-I mRNA was detected in all tissues and with quantitative RT-PCR, and no significant changes were detected until placentation. Immunohistologically, at the apex and corpus site, the average number of MHC-II positive cells increased from the pre-implantation to the post-implantation stage (apex: 1.54 ± 1.21 to 3.82 ± 2.93; corpus: 1.62 ± 1.9 to 5.04 ± 4.95; p < 0.05). The greatest numbers of MHC-II positive cells were observed at placentation sites (6.64 ± 5.9). In parallel, a marked increase in the relative mRNA expression of MHC-II in uterine tissues was assessed from the pre-implantation to the placentation stage (relative to Glycerinaldehyd-3-phosphate-Dehydrogenase (GAPDH): 6.9 ± 9.5, 8.4 ± 5.8, p > 0.05). Immunohistologically, in the salpinx, significantly greater numbers of MHC-II positive cells were found in the tissues of pregnant animals than in the control group (p < 0.05). It is proposed that the increase in MHC-II is pregnancy-related, even though the impact on maintenance of canine pregnancy is still unclear.