Limited contribution of circulating cells to the development and maintenance of nonhematopoietic bovine tissues.

Bone marrow-derived stem cells appear surprisingly multipotent in experimental settings, but the physiological significance of such plasticity is unclear. We have used sex-mismatched cattle twins with stably chimeric hematopoietic systems to investigate the general extent of integration of circulating cells to the nonhematopoietic cell lineages in an unmanipulated large mammal. The donor-derived (Y+) nonhematopoietic cells in female recipient tissues were visualized by Y-chromosome specific in situ hybridization combined with pan-leukocyte labeling. Y+ leukocytes were frequent in all tissues, but in 11 of 12 animals, average contribution to nonhematopoietic lineages was in any tissue below 1% (in brain <0.001%). Significantly higher integration rate was detected in regenerating granulation tissue. Also, one animal showed a high frequency of nonhematopoietic Y+ cells in several tissues, including intestinal epithelium and mammary gland stroma. In conclusion, circulating cells do not appear significant in the development and maintenance of nonhematopoietic bovine tissues, but may be important in regeneration and other special conditions.

Age dependent changes in plasma anti-Müllerian hormone concentrations in the bovine male, female, and freemartin from birth to puberty: relationship between testosterone production and influence on sex differentiation.

To understand the behaviour of the gonads, in terms of hormonal secretion, in a model of intersexual development naturally occurring in mammals, we determined plasma concentrations of testosterone, progesterone, and anti-Müllerian hormone (AMH) in bovine freemartins, and compared them to normal levels measured in males and females from birth to puberty. We found that newborn males and freemartins have very high concentrations of AMH (over 700ng/ml). Conversely, plasma AMH concentration is always below 120ng/ml in females. While values remain stable in males for the first five months of life, they sharply decrease in the freemartins within the first fortnight, and reach female levels, which demonstrates that AMH is essentially originated in the male twin. In young bulls the trend of plasma testosterone concentrations is opposite to that of the AMH. The rise in testosterone production at puberty corresponds to a sharp decline in AMH concentrations. Bovine plasma concentrations of AMH are surprisingly higher than those measured in other mammals, including man and mouse. The results obtained are discussed in reference to comparative aspects of endocrine functions.

Inter-relationships among gonadotrophins, reproductive steroids and inhibin in freemartin ewes.

Freemartins are sterile XX/XY chimaeras that occur as a result of placental fusion between male and female fetuses during early pregnancy. Freemartins occur predominantly in cattle, although the prevalence of ovine freemartinism is increasing. In this study, the reproductive endocrinology of ovine freemartins was compared with that of normal sheep. Freemartins had significantly (P < 0.001) higher basal concentrations of LH and FSH than did normal ewes or rams, although the response of LH to GnRH (10 microg) was similar in freemartins, ewes and rams. Resting concentrations of oestradiol were similar in freemartins and ewes and were increased in both after eCG administration. Testosterone concentrations were higher in freemartins than in ewes, but were unresponsive to GnRH or eCG. Administration of 62.5 mg progesterone or 25 lg oestradiol twice a day for 3 days suppressed LH concentrations to baseline values in freemartins, ewes and rams. In ewes, 500 microg oestradiol administered twice a day caused preovulatory surges in LH concentrations, but suppressed LH in freemartins to baseline values. Thus, LH secretion can potentially be regulated in freemartins by gonadal steroids. FSH concentrations in freemartins were not suppressed by doses of inhibin that were effective in ewes and rams. Therefore, freemartins behave in part like castrated animals, as they have high basal concentrations of LH and FSH, which can be stimulated by GnRH and suppressed by gonadal steroids. Conversely, inhibin does not suppress FSH concentrations in freemartins, and freemartins have circulating concentrations of steroids intermediate between those of castrated and normal animals.

Birth of a Holstein freemartin calf co-twinned to a schistosomus reflexus fetus.

An unusual case of a live Holstein freemartin calf co-twinned with schistosomus reflexus fetus is presented here. Delivery of the schistosomus reflexus was achieved by fetotomy 24 h after manual delivery of a live heifer calf. The dam subsequently experienced concurrent metritis and left displacement of the abomasum; however, she conceived following insemination 85 d post partum. Cytogenetic examination of the live heifer using lymphocyte culture and cutaneous fibroblast cell culture failed to demonstrate chromosomal chimerism, whereas polymerase chain reaction (PCR) detected the presence of the bovine Y-chromosome marker BRY-1. Low concentrations of testosterone, estradiol and progesterone at 3, 6, 24 and 48 h after administration of hCG were detected in the serum of the freemartin heifer. Genetic, hormonal, histological and clinical findings established the live female co-twin calf was a freemartin, an abnormality of phenotypic sex. Failure to detect any significant peripheral concentrations of androgen supports the hypothesis that masculinization of the freemartin reproductive tract arises from diffusion of androgen and possibly other substances from the male co-twin rather than from endogenous production of androgen within the freemartin. This report documents that the freemartin condition can be induced by a male fetus with severe developmental abnormalities.

The possibility of early estimation for fertility in bovine heterosexual twin females.

To diagnose the possibility of early estimation for fertility in bovine heterosexual twin females, we designed a new diagnostic program. The 9 freemartins (FM) and 5 normal females (Normal) were used in this study. All 14 cases, at 4 months of age, were given Pregnant Mare Serum Gonadotrophin (PMSG) and human Chorionic Gonadotrophin (hCG) 1.5-2 days later. Thereafter, the concentration of estradiol-17 beta (E2) was determined by RIA, and that of progesterone (P) was done by RIA and EIA (Ovcheck EIA Kit). The concentration of E2 in the Group of Normal rapidly increased after administration of PMSG, but in the Group of FM, the concentration of E2 changed in very low levels over 14 days. The concentration of P in the Group of Normal rapidly increased after administration of PMSG, but in the Group of FM, the concentration of P changed in very low levels over 14 days.

[Comparison of different methods for the diagnosis of freemartinism--blood group serology, cytology and polymerase chain reaction]. / Vergleich verschiedener Methoden zur Diagnose des bovinen Freemartinismus--Blutgruppenserologie, Zytologie und Polymerase-Ketten-Reaktion.

The percentage of freemartins among blood samples tested by chromosome analysis amounted to 83.9%, by blood group serologie 71.4%. 126 blood samples have been tested by blood group serology and PCR. Employing blood group serology, 71.3% and using PCR with BOV97M primers 85.8% of the animals proved to be freemartins. 40 blood samples were additionally analysed using PCR with zinc-finger-gene primers. 36 animals (90%) were identified as being freemartins by means of BOV97M and 34 animals (85%) by means of the zinc-finger-gene primer. The PCR method proved to be a rapid and very sensitive method for the diagnosis of freemartins and also suitable for routine testing. The BOV97M primer showed to have a higher Y chromosome specificity than the zinc-finger-gene primer.

A sensitive radioimmunoassay for bovine anti-Müllerian hormone, allowing its detection in male and freemartin fetal serum.

Two monoclonal antibodies have been used to set up a solid-phase RIA for bovine anti-Müllerian hormone (bAMH). One AMH unit is defined as the amount released by 1 g of bovine fetal testicular tissue during a 4 h incubation period. Calibration curves were prepared using aliquots of a standard 500 ml pool of incubation medium, containing 200 AMH mU/ml, diluted either in 50% pig testes incubation medium, 5% horse serum, 10% female calf fetal serum or pure female calf fetal serum. Linearization of the calibration curves was achieved through "logit-log" transformation, all four lines were parallel. Within and between-assay variability were less than 5%. The RIA is at least 600 times more sensitive than the bioassay for anti-Müllerian activity and can detect AMH in male and freemartin fetal serum.

Changes in serum FSH concentrations in the pig during development.

Serum FSH concentrations were measured in fetal and prepubertal pigs between 40 days postcoitum and 25 weeks after birth. In addition, serum FSH was estimated in prepubertal, unilaterally cryptorchid, freemartin and castrated pigs. The average serum FSH concentrations in male and female fetuses was low (less than 2 ng/ml) until 80 days p.c. During the remaining fetal period, concentrations in females were elevated (7.9 +/- 0.4 ng/ml) and remained fairly constant after birth (16.3 +/- 0.8 ng/ml). In the male, serum FSH concentrations gradually rose to 22.5 +/- 5.5 ng/ml during the first 3 weeks after birth and declined thereafter. The changes in FSH concentrations in male pigs are reflected in gonadal-development. In contrast, in fetal and prepubertal females, ovarian development seems not to be influenced by changes in serum FSH concentrations. Unilateral cryptorchidism did not affect serum FSH concentrations. After castration, however, concentrations rose significantly. In freemartin pigs concentrations were similar to those in female pigs.

The acute effects of oestradiol-17beta and synthetic LH-RH on plasma LH levels in freemartin heifers.

Injection of oestradiol was followed by a surge of plasma LH within 24 h in only 7 of 12 freemartins. Elevations of plasma LH were less than those reported for normal non-cyclic heifers, but some freemartins showed a delayed, or more prolonged, LH response. Responsiveness to oestradiol was not related to degree of chimaerism or plasma androstenedione level, and most of the animals responded similarly in two trials carried out 4 months apart, during which time plasma androstenedione levels had more than doubled. Freemartins which showed an LH surge after oestradiol treatment released greater amounts of LH after the injection of LH-RH than did non-responders.

The acute effect of oestrogen injection on plasma LH in freemartin heifers.

The acute effects on plasma LH concentrations of an injection of oestradiol-17beta were studied in 7 non-cyclic heifers and 19 freemartins. One freemartin showed a normal LH surge due to the positive feedback effect of oestrogen on the hypothalamus. Of the other 18 freemartins, 4 showed positive increases in plasma LH and 6 were unclassified. There was no correlation between the degree of chimaerism and responsiveness to oestrogen. The results also showed that injected oestradiol suppressed the spontaneous fluctuations of plasma LH.

Plasma progesterone, androstenedione and testosterone concentrations in freemartin heifers.

Plasma concentrations of testosterone, androstenedione and progesterone in freemartins, and normal cyclic and non-cyclic heifers were studied. The plasma testosterone concentrations were in general less than 10 pg/ml in all animals. The mean androstenedione concentration of 28 pg/ml in 10- to 12-month-old freemartins was significantly lower than the mean of 58 to 60 pg/ml for normal 10- to 12-month-old heifers. At 24 months of age the mean androstenedione concentration in the freemartins had risen significantly to 65 pg/ml.