Characterization of immunoreactive IGF-I pattern during the peri-ovulatory period of the oestrous cycle of thoroughbred mares and its relation to other hormones.

The aim of this study was to characterize ir-IGF-I pattern and its relation to other hormones during the oestrous cycle in mares. Nine non-pregnant non-lactating pluriparous thoroughbred mares were used. The studied mares were examined ultrasonically and bled daily to follow the ovarian changes and the hormonal milieu for a complete Interovulatory interval (IOI). Two (minor and major) follicular waves were characterized per IOI in thoroughbred mares. The largest follicle of the first follicular wave (DF1) was firstly detected at D - 1.75 ± 0.47 with a growth rate of 2.78 ± 0.14mm/day and maximum diameter of 22.45 ± 0.75mm on day 6.65 ± 0.82. The largest follicle of the second follicular wave (DF2) had a growth rate of 2.15 ± 0.29 mm/day, reached a maximum diameter of 42.70 ± 2.63 mm on D 19.25 ± 0.43. Ir-IGF-I increased significantly prior to ovulation and had a similar pattern to oestrogen (r = 0.84, p < 0.05), suggesting that the ovarian follicles are the main source of circulating ir-IGF-I during the oestrous cycle of mares and that ir-IGF-I may be a crucial factor in follicular differentiation and maturation. In conclusion, this study demonstrated that ir-IGF-I is secreted during the oestrous phase of the cycle concomitant with the development of the future ovulatory dominant follicle, and it may act in synergy with other hormones for the selection and differentiation of the dominant follicle.

Effect of a single intrauterine administration of recombinant bovine interferon-τ on day 7 of the estrous cycle on the luteal phase length and blood profile in dairy cows.

This study tested the effect of recombinant bovine interferon-tau (rboIFN-τ) on the length of estrous cycle, luteal lifespan and side effects of rboIFN-τ in the cow. A normal estrous cycle in six non-lactating cycling Holstein cows was observed (non-treated cycle), and either 2.0 mg of liposomalized rboIFN-τ (treated cycle) or bovine serum albumin (BSA; placebo cycle) was infused in the uterus on day 7 of the estrous cycle (day 0=day of ovulation). Rectal temperature, heart rate and respiratory rate were recorded and blood samples were collected before and after the treatments. The length of the estrous cycle and corpus luteum lifespan in rboIFN-τ treated cycles were not significantly different from those of the non-treated and placebo cycles. In contrast, the rboIFN-τ treatment caused a transient increase in rectal temperature and a decrease in the number of peripheral lymphocytes and neutrophils after the treatment.

Computed tomography of the prostate gland in apparently healthy entire dogs.

OBJECTIVES: To describe the computed tomography (CT) features of the prostate gland and determine prostate size using CT in entire male dogs. METHODS: The prostate gland was evaluated in 35 dogs. Morphological features including homogeneity, delineation, shape and intraprostatic differentiation were assessed. Height, length, width, area, volume and attenuation values of the prostate gland were measured. Ratios of prostatic height (rH), length and width to the sixth lumbar vertebral body length were calculated. Relationships of prostatic dimensions with body weight and age were evaluated. RESULTS: The prostate gland was homogeneous in 29 dogs on non-contrast images and 18 of 24 dogs on postcontrast images. Transverse images revealed a semi-oval prostate gland in 29 dogs and irregularly shaped prostate gland in 6 dogs. A prominent median septum was observed in postcontrast images. Significant positive correlations were found between body weight and age and all prostatic dimensions except between age and rH. The mean ± sd values for attenuation were 59·3 ± 9·1 and 121·3 ± 22·7 HU in non-contrast and postcontrast image, respectively. CLINICAL SIGNIFICANCE: CT can be useful for evaluating morphological features of the prostate gland. Prostatic length or width is a better measure than height for computed tomographic estimation of prostate size.

Ovarian stimulation with follicle-stimulating hormone under increasing or minimal concentration of progesterone in dairy cows.

The objective of this study was to investigate the effect of the presence or absence of Corpus luteum (CL) on the follicular population during superstimulation in dairy cows (Holstein-Friesian cattle). Animals were divided into two groups as follows: (1) Growing CL group (G1): Cows (n=7) received a total dose of 28 Armour units (AU) follicle-stimulating hormone (FSH) through the first 4 d (twice daily) after spontaneous ovulation (Day 0). (2) CL Absence group (G2): Cows (n=10) received prostaglandin F(2alpha) (PGF(2alpha)) at 9 or 10 d after ovulation. After 36h, all the follicles (larger than 5mm) were aspirated (Day 0). The FSH treatment started 24h after aspiration and continued for 4 d. The number of small (3 to <5mm), medium (5 to <8mm), and large (> or = 8mm) follicles was examined on Days 1, 3, and 5 in all groups. Blood samples were collected daily for 5 d, and progesterone (P(4)), estradiol (E(2)), insulin-like growth factor-1 (IGF-1), and growth hormone (GH) in plasma were measured by enzyme immunoassays. The results showed that in G1, the P(4) level increased gradually from 0.5 ng/mL at Day 1 to 2 ng/mL at Day 5, whereas in G2, the P(4) level was completely below 0.5 ng/mL. All cows of the G2 group showed an increase of E(2) at Day 3 or Day 4 followed by an increase of IGF-1 within 24h, while GH increased concomitantly with the E(2) increase in 8 of 10 trials. On the other hand, cows of the G1 group showed neither E(2) nor IGF-1 increase. Moreover, at the end of the treatment, the number of follicles in the G2 group was significantly increased compared with that of the G1 group (22.8+/-2.0 vs. 11.6+/-2.0). In conclusion, low P(4) level during FSH treatment enhanced multiple follicular growth and E(2) secretion, which was followed by increase of IGF-1 and GH. Therefore, the absence of the CL may play a critical role in the superovulation response by controlling the number of growing follicles.

Population of follicles and luteal structures during the oestrous cycle of mares detected by three-dimensional internal structure microscopy.

The structure of the equine ovary is different from that of other mammals in its extremely large size, the presence of ovarian fossa and the inverted location of its cortex and medulla. A three-dimensional internal structure microscopy (3D-ISM), which consists of a computer-controlled slicer, a CCD camera, a laser disc recorder and a PC, is very useful for the observation of the internal structures in equine ovaries. In addition, the three-dimensional images of follicles and corpus luteum (CL) reconstructed by the segmentation technique can clarify the spatial arrangement in the equine ovary. In this study, to understand the changes in the ovarian internal structures of the mare during the oestrous cycle, the size and numbers of follicles and luteal structures were analysed by 3D-ISM in addition to the concentrations of progesterone (P(4)) and oestradiol-17beta. As a result, many small follicles (<10 mm in diameter) were detected. It was recognized that the luteal structures were distinguished into three types, such as the corpus haemorragicum (CH), which is formed by blood elements at the cavity after ovulation, CL and corpus albican (CA). There were some CHs and CL in the group, which had the concentration of P(4) > 1 ng/ml. CHs were also observed in the group, which had low level of P(4) (P(4) < 1 ng/ml). CAs were found regardless of the P(4) level. In conclusion, 3D-ISM enabled the internal observation of the ovarian structures in detail, and estimation of the stage of the ovarian cycle with complementary physiological information. The findings by 3D-ISM provide basic information for clinical applications.

Short communication: A positive relationship between the first ovulation postpartum and the increasing ratio of milk yield in the first part of lactation in dairy cows.

The aim of the present study was to examine the relationship between characteristics of the lactation curve, on the basis of daily milk yield, and ovulation within 3 wk postpartum as an indicator of early return to luteal activity in dairy cows. Lactation records from 46 lactating Holstein cows between calving and 305 d postpartum were studied. Milk samples were collected twice weekly between d 7 and 100 for later determination of progesterone concentrations. Occurrence of an early first ovulation was determined by an increase in milk progesterone by 3 wk after calving. Milk yield was recorded daily until 305 d postpartum, and average yield was calculated weekly. The lactation curve was characterized by 8 indices on the basis of the weekly average of milk yield as follows: a) first-week milk yield; b) peak milk yield; c) actual 305-d milk yield; d) peak week; e) difference in milk yield between the first week and peak week; f) difference in milk yield between the peak week and last week (43rd week postpartum); g) ratio of increase in milk yield between wk 1 and the week of peak yield; and h) ratio of decline in milk yield between the week of peak yield and the last week. Indices g and h were calculated as linear. The number of cows having ovulated by 3 wk postpartum was 22 (47.8%). The resumption of ovarian cycles with normal luteal phases occurred earlier in ovular cows than in anovular cows (32.0 d vs. 57.1 d). Although total milk yield did not differ between ovular and anovular cows, the ratio of increase in milk yield from the first week to the peak week (index g) in ovular cows was smaller compared with that of anovular cows (1.71 vs. 2.54). In addition, the ratio of increase in milk yield from the first week to the third week postpartum was greater in anovular cows by 3 wk postpartum (ovular = 1.43 +/- 0.23 vs. anovular = 2.32 +/- 0.29). In conclusion, the present study demonstrates that a greater increasing ratio of milk yield during early lactation may delay resumption of ovarian cycles after parturition. Therefore, this study is the first to demonstrate statistically that a smaller increasing ratio of milk yield (index g) during early lactation may have a beneficial effect on the first ovulation by 3 wk postpartum.

Study of early pregnancy factor (EPF) in equine (Equus caballus).

PROBLEM: Early pregnancy factor (EPF) is an immunosuppressive protein detected in the early pregnancy serum. We have already reported that we developed the rosette inhibition test for mare EPF and detected EPF in thoroughbreds. The aim of this study was to determine whether or not our method could be used clinically. METHODS OF STUDY: The rosette inhibition test for equine EPF was carried out on serum from six nonpregnant and six pregnant Shetland ponies, a female and a male Chinese pony, and four nonpregnant and 13 pregnant thoroughbred mares. In the thoroughbreds sera were collected during the pregnancy period. Furthermore, we measured progesterone and detected pregnant mare serum gonadotrophin (PMSG) in order to confirm pregnancy of the Chinese pony 3 and 6 months after mating. RESULTS: In the nonpregnant Shetland ponies, the rosette inhibition titre (RIT) was 6.0+/-1.0 and EPF was negative. In contrast, in the pregnant ponies, the RIT was 9.2+/-0.4 and EPF was positive. Based on these results, we diagnosed pregnancy of the Chinese pony. The RIT of the female Chinese pony (3 months after mating) was above 10 and EPF was positive. Furthermore, we detected PMSG and progesterone in the serum of this pony. EPF appeared in the maternal blood circulation at 24-72 hr after mating, it was detected until the second trimester, and after that it disappeared from the maternal serum. CONCLUSIONS: The pony's EPF was detected by using the same rosette inhibition test as in the thoroughbred and was present from 24 to 72 hr after mating until the second trimester. The results indicated that our method was useful for pregnancy diagnosis of Equine.

Inhibin secretion in the stallion.

To examine the physiological role of inhibin in the stallion, a heterologous radioimmunoassay (RIA) based on a bovine RIA was validated and used to measure immunoreactive (ir)-inhibin concentrations in plasma and testicular homogenates. The bioactivity of equine testicular inhibin was also examined using an assay for suppression of FSH secretion from rat anterior pituitary cells. In addition, to identify the cell responsible for secreting testicular inhibin, the localisation of inhibin in the testis was investigated by an immunohistochemical method using a polyclonal antibody against (Tyr30)-porcine inhibin alpha(1-30) NH2. In the RIA, parallel dose response curves were obtained for the bovine inhibin standard and serial dilutions of stallion plasma and equine testicular homogenates. Parallel FSH inhibition curves were also observed for the bovine inhibin standard and serial dilutions of equine testicular homogenates in the bioassay. The inhibition of FSH secretion from rat pituitary cells by equine testicular homogenates was neutralised by an antiserum against bovine inhibin in vitro. Plasma concentrations of ir-inhibin, testosterone and oestradiol-17beta in stallions decreased abruptly after bilateral gonadectomy and FSH and LH concentrations in the plasma subsequently increased. Therefore, circulating inhibin in the stallion appeared to be largely of testicular origin. The histochemical results showed for the first time that strong immunopositive staining for inhibin occurred in the Leydig cells of the testes. Sertoli cells were also stained by the inhibin antibody but the reaction was weaker than that in Leydig cells. These results indicate clearly that both Leydig and Sertoli cells are potential sources of testicular inhibin in the stallion. A clear increase in plasma ir-inhibin concentrations was observed during the natural breeding season. Similar seasonal changes in the plasma concentrations of testicular steroid hormones and pituitary gonadotrophins occurred throughout the year. In conclusion, the testes appear to be the main source of inhibin, and testicular inhibin is secreted by Leydig and Sertoli cells in stallions. The positive correlations between plasma ir-inhibin and testicular activity during both the breeding and nonbreeding seasons indicate that plasma ir-inhibin is a useful indicator of reproductive activity in the stallion.

Lipid peroxide levels and superoxide-scavenging abilities of Sera obtained from hotbred (Thoroughbred) horses.

Hotbred (Thoroughbred) horses were grouped into three classes according to the levels of constant physical exercise (foals, 6 months old; racing horses, 5 years old; horses for breeding, 6-10 years old), and lipid peroxide levels in their sera were measured as thiobarbituric acid-reactive substances. No significant differences were observed among them. The superoxide-scavenging abilities of sera were measured next; to examine the antioxidative properties of hotbreds, and were found to be highest in the racing horses. The higher scavenging ability of the racing horses might contribute to keep their lipid peroxide levels as low as those of the other two groups. HPLC analysis of substances in sera suggested that the presence of albumin-bound bilirubin was one of the reasons for the high superoxide-scavenging ability of sera of the racing horses. When the hotbreds were compared with coldbred (crossbred) horses, the lipid peroxide levels of hotbreds was higher (7.0 +/- 1.2 nmol/ml) than that of coldbreds (2.6 +/- 0.7 nmol/ml). Comparison of the superoxide-scavenging abilities of sera between hotbreds and coldbreds showed that the hotbreds possessed higher scavenging ability than the coldbreds. These results indicated that the lipid peroxide level in sera of hotbreds was higher than that of coldbreds regardless of the higher superoxide-scavenging abilities of sera.

Deletion of the SRY region on the Y chromosome detected in bovine gonadal hypoplasia (XY female) by PCR.

In 6 sterile heifers which were female in appearance and in genital organs, all tissues derived from blood, skin, spleen and kidney had a single 60,XY type of metaphase plate. Although the SRY gene was detected by PCR in normal bull, it was not detected in normal cow or in 3 60,XY cases studied.

A fast, convenient diagnosis of the bovine freemartin syndrome using polymerase chain reaction.

To establish the polymerase chain reaction (PCR) method for detecting the XY cells in cases suspected to have the bovine freemartin syndrome, a PCR reaction test was conducted on blood from a normal bull diluted in blood from a normal cow. From the results obtained, it was shown that the Y-specific sequence was detectable down to a concentration of 0.1%. Various types of the bovine freemartin syndrome, which occurs in heterosexual twins, single-born sterile heifers, and heifers born with Acardius amorphus, were examined by the chromosome analysis and the PCR method. The Y-specific sequence was detected in all 26 cases that showed chromosome chimerism but which was absent in the 5 cases without a chimerism. The PCR method was found to be effective and convenient for quickly diagnosing the various types of bovine freemartin syndrome.

Minute fragment observed in a bovine pedigree with Robertsonian translocation.

A minute fragment was found in the metaphase plates obtained from one Holstein bull carrying Robertsonian translocation. The pedigree investigation indicated that three out of 34 offspring had the same minute fragment as their sire. Fluorescence in situ hybridization (FISH) studies with bovine satellite DNA revealed that the minute fragment was hybridized with this DNA and was part of the bovine centromere. We propose that the minute fragment is the product of centric fusion and is transmitted to offspring without being lost in the subsequent cell divisions.

Chromosomal pericentric inversion detected in a sow and her piglets.

Forty-four pigs with the suspicious symptoms of porcine stress syndrome (PSS) were selected for chromosome analysis. Cytogenetic evaluation by means of the G-banding technique revealed that one sow had an abnormal (38,XX, inv (1p+q-) (2.1;1.1) karyotype. The same abnormality was also detected in 8 of 13 offspring of this sow. However, there was no correlation between the chromosome abnormality and PSS. The chromosome abnormality did not give rise to a reduction in the fertility of this sow or in the viability of her offspring. This case represents the first reported instance of pericentric inversion in swine.

Sperm chromosome study of two bulls heterozygous for different Robertsonian translocations.

Haploid chromosomes of a total of 354 spermatozoa from two bulls heterozygous for different Robertsonian translocations, a Holstein-Friesian bull carrying a t(1;21) and a Japanese Black bull carrying a t(7;21), were analyzed using an interspecific in vitro fertilization system with zona-free hamster oocytes. The proportion of chromosomally normal and balanced spermatozoa was approximately equal in both carriers (51.8% and 47.0% in the 1/21 carrier, and 47.3% and 50.0% in the 7/21 carrier). The combined incidences of normal and balanced spermatozoa, i.e., incidences of spermatozoa resulting from alternate meiotic segregation were very high (98.8% and 97.3%) in both carrier. On the contrary, the incidences of chromosomally unbalanced spermatozoa resulting from adjacent meiotic segregation were only 0.6% and 2.7%. These results indicate that the alternate segregation of a trivalent chromosome is predominant in these Robertsonian translocation carriers.

Serum esterase genetics: identification and hormone induction of the Es-1b esterase in inbred rats.

A previously unrecognized esterase from the sera of the appropriate strains of the rat Rattus norvegicus was revealed by a discontinuous polyacrylamide gel electrophoretic technique. This esterase migrated in the albumin region, whereas a previously known major albumin esterase controlled by the Es-2 locus migrated in the postalbumin region when the method was used. The new albumin esterase component which separated from the Es-2 esterase was identified as the product of the Es-1b gene. The new albumin esterase was not detectable in the sera of sexually mature males of the appropriate genotype, because the activity level of this esterase was influenced by sex hormones, especially androgen.