Ultrasonographic examination of postpartum uterine involution in sows.

Physiological uterine involution during the puerperium period is essential for sow reproductive health. Uterine involution in sows has mainly been described using macroscopic and histological examination after slaughter. The aim of this study, therefore, was to describe the continuous regression of uterine diameter from day 2-14 after parturition and on the day before weaning using ultrasonography in sows housed in a free farrowing system and in farrowing crates. Diameter of three uterine cross-sections was measured at 24 -hs intervals in 46 sows housed in a free farrowing system in Switzerland and 49 sows housed in farrowing crates in Germany. Overall, there was continuous regression of uterine diameter during the lactation period in both groups. Median diameter of the uterus decreased from 32.4 mm (min: 18.6 mm, max: 52.3 mm) on day 2-9.0 mm (min: 7.6 mm, max: 12.7 mm) on the day before weaning (on average 30 days p.p.) in sows housed in free farrowing systems. Median diameter of the uterus of sows in farrowing crates decreased from 38.5 mm on day 2 (min: 21.6 mm, max: 56.3 mm) to 10.1 mm (min: 8.8 mm, max: 13.6 mm) the day before weaning (on average 29 days p.p.). Interestingly, parity, obstetrical intervention and administration of oxytocin was not associated with the decrease of uterine diameter during the study period. In summary, the ultrasonic examination is a suitable method to evaluate diameter of the uterus during the puerperium period in sows and to describe uterine involution in vivo.

Prolonged parturition and impaired placenta expulsion increase the risk of postpartum metritis and delay uterine involution in sows.

It was hypothesized that prolonged parturition and impaired placenta expulsion increase the risk of postpartum metritis and delay uterine involution. At parturition, for 99 Yorkshire x Large White sows (parity 2-5), we determined the number of liveborn (NLP; 14.8 ± 3.4) and stillborn piglets (NSP; 1.1 ± 1.1), farrowing duration (FAR, time between first and last piglet; 333 ± 249 min), placenta expulsion duration (PLA, time between first and last placental part; 292 ± 241 min) and number of expelled placental parts (PART; 3.0 ± 1.0). FAR was categorized as 'normal' (<300 min; n = 44/99) or 'prolonged' (>300 min; n = 55/99). The relative PLA (rPLA; (PLA * 100)/FAR; 76 ± 101%) and the relative PART (rPART; (PART * 100)/(NLP + NSP); 22 ± 8%) were calculated and placenta expulsion was categorized as 'normal' (rPLA and rPART > 10%; n = 93/99) or 'impaired' (relPLA and relPART < 10%; n = 6/99). We also recorded whether manual palpation occurred (Yes/No) and/or oxytocin was used (Yes/No). After parturition, an ultrasound examination of the uterus was performed once for each sow between the 2nd and 7th day postpartum and the uterine size (mean sectional area of three to five uterine cross-sections) and intrauterine fluid accumulation (Yes/No) were recorded. Uterine size was categorized as 'normal' (n = 55/99) or 'enlarged' (n = 44/99) and used as an indicator of delayed uterine involution. Intrauterine fluid was used as an indicator of metritis. Prolonged FAR (35/54, 2.0 ± 0.5, 13.1, 7.6; n/N, ß ± SE, Wald χ2, Odds; P = 0.001), manual palpation (12/17, 1.5 ± 0.7, 4.4, 4.3; P = 0.036), oxytocin administration (18/31, - 1.5 ± 0.7, 4.7, 0.2, P = 0.040) and NSP ≥ 2 (15/21, 1.4 ± 0.7, 3.8, 3.9; P = 0.052) were associated with increased uterine size (n = 44/99) and NSP ≥ 2 (7/21, 2.6 ± 0.9, 8.7, 13.7; P = 0.003), manual palpation (10/17, 1.8 ± 0.8, 5.0, 6.0; P = 0.025), prolonged FAR (13/15, 1.7 ± 0.8, 4.3, 5.7; P = 0.039) and impaired PLA (4/6, 3.3 ± 1.7, 4.0, 26.9; P = 0.044) with intrauterine fluid accumulation (n = 15/99). The results confirm the hypothesis, indicating that prolonged parturition and impaired placenta expulsion increased the risk for postpartum metritis. Stillborn piglets and manual palpation were also identified as risk factors. Postpartum metritis delays uterine involution whereas the use of exogenous oxytocin supports it.

Development of Deep and Upper Neuronal Layers in the Domestic Cat, Sheep and Pig Neocortex.

The neocortex plays a key role in cognition, volitional motor control and sensory perception and has undergone tremendous expansion during evolution. The mature neocortex consists of radially aligned neurons that are arranged in six layers. Layers II-VI are often split into two groups: deep and upper layers, both building up the so-called cortical plate during embryonic and foetal development. So far cortical neurogenesis, including the generation of deep and upper layers, has mostly been studied in laboratory rodents and primates. However, precise data for most companion animals are lacking. This study determined the main period of neurogenesis, specifically the timing of deep and upper layer generation, in the developing domestic cat, pig and sheep neocortex using immunohistochemistry for specific neuronal markers, that is Tbr1 and Brn2. We found that the general sequence of neural events is preserved among cat, pig, sheep and other mammalian species. However, we observed differences in the timing of the overall cortical neurogenic period and occurrence of distinct neural events when these three species were compared. Moreover, our data provide further evidence that the cortical neurogenic period and gestation length might be tightly related. Together, these data expand our current understanding of neocortex development and are important for future studies investigating neocortex development and expansion especially in companion animals.

[Uterine neoplasia in two geriatric miniature pet pigs - diagnostics and therapy. Two case reports]. / Uterustumore bei älteren Miniaturschweinen - Diagnostik und Therapie. Zwei Fallberichte.

Miniature pet pigs are becoming ever more common among the patients of small animal practices. Due to an advanced age attained in these animals in comparison with conventional pigs in pork production, miniature pet pigs develop age-related diseases that are infrequently observed in farm pigs, including neoplastic alterations of the uterus. We describe two cases of a uterine adenocarcinoma and a leiomyoma, respectively, in two miniature pet pig sows. Options and limits of diagnostic measures (unspecific clinical symptoms, x-ray, sonography and computed tomography) and therapy within the legal limits as well as the risk of an incorrect diagnosis based upon the more common differential diagnoses in swine medicine, including obstipation, cystitis and endometritis, are discussed.

Advances in Breeding Management and Use of Ovulation Induction for Fixed-time AI.

The objective of the breeding herd is the predictable and consistent production of high quality pigs. To achieve this objective, an appropriate number of females need to be mated in each breeding week and they should maintain their pregnancy and deliver large litters. Many factors can impact achievement of optimal sow productivity, particularly breeding management. Most matings will involve artificial insemination (AI), and successful AI requires deposition into the cervix (or beyond) of sufficient viable high quality sperm at an appropriate time relative to ovulation. This is facilitated by improved knowledge of the sow's ovarian function prior to and during her oestrous period. Realization of the importance of establishing an adequate sperm reservoir in the oviduct at an appropriate time relative to ovulation has led to advances in the management of AI. The future of AI will likely involve insemination of single doses of high genetic merit semen, potentially having a reduced sperm concentration which is made possible by knowledge of the effect of site of sperm deposition on sow fertility. In particular, knowledge of when a sow is likely to ovulate during a natural or induced oestrous period will prove invaluable in the maintenance of herd productivity. This review will examine options for breeding management, including the control of oestrus and ovulation, on sow herd reproductive performance.

[Reproductive disorders in the female pig: causes, manifestation, diagnostics and approach in herd health care]. / Fertilitätsstörungen beim weiblichen Schwein. Ursachen, Manifestation, Diagnostik und Vorgehen im Rahmen der tierärztlichen Bestandsbetreuung.

Reproductive failure is the most common cause for culling sows from production herds. These failures can affect individual pigs, but can also be a farm problem. Any region of the pig's genital tract can be diseased, with the uterus being the most frequently affected organ. Numerous microorganisms and toxins are known to cause genital pathology in the sow. Although reproduction problems are considered to be multifactorial, recognizing genital pathology is crucial as the first step in the diagnostic workup of reproductive disorders. This contribution briefly reviews the most common genital disorders in the female pig with respect to incidence, pathogenesis and clinical picture. In a second part, the gynaecological examination and the steps in the diagnostic workup are presented.

Establishment of a practical sperm penetration test for bovine semen.

OBJECTIVE: Several methods for evaluating semen quality have been developed in addition to traditional semen analysis. Most of these methods are very complex and require expensive technical equipment as well as special knowledge to be performed. The aim of this study was to establish an easy and cost-effective sperm penetration test for bovine semen using artificial media. Additionally a standard procedure for test performance should be figured out. MATERIAL AND METHODS: In the first part of the study four different capillary tubes or pipettes were evaluated in order to determine the most appropriate one to use in the penetration test. In the second part of the study the tubes were filled with different compositions of polyacrylamide gel and subsequently incubated at 37.5 °C for 30 minutes. Sperm penetration was then evaluated using a phase contrast microscope, measuring the penetration distance of the first, the first motile and the five vanguard motile spermatozoa as well as determining the number of sperm cells cumulated per low power field (LPF) at three distinct distances in the tube. RESULTS: Non-heparinised haematocrit capillary tubes were the most appropriate tubes for performance of sperm penetration test as 100% of used capillaries were evaluable after incubation and size of the capillaries allowed a simple handling and good visualisation of migrated spermatozoa. Best results were achieved with 1.9% polyacrylamide gel containing phosphate-buffered saline. Measuring the penetration distance of the vanguard motile sperm cell can be recommended for test evaluation. CONCLUSION: A sperm penetration test with 1.9% polyacrylamide using non-heparinised capillary tubes was established. Results showed high reproducibility, the procedure is simple to carry out and only requires a phase contrast microscope.

[Physiology and pathology of reproduction in domesticated New World camelids with special emphasis on ultrasonography]. / Physiologie und Pathologie der Fortpflanzung bei weiblichen, domestizierten Neuweltkameliden. Übersicht mit besonderer Berücksichtigung der ultrasonographischen Diagnostik.

The number of New World camelids in Germany is increasing. Owners and breeders are usually well educated regarding their animals. For practitioners, this means being up-to-date with respect to their veterinary knowledge. This includes the physiology and pathology of reproduction. Specifics of reproduction in domesticated New World camelids are an induced ovulation, the absence of cyclic sexual activity, a relatively long gestation of 336-349 days and a predominantly left-horn gestation. Ultrasonography plays an important role as part of the gynecological examination. Generally, the ultrasonographic examination can be performed transrectally and transcutaneously in the left or right flanks. Transrectal ultrasonography has to be carried out with particular caution to avoid rectal injuries. An accurate pregnancy diagnosis by transrectal scanning is possible starting from day 20 of pregnancy; using transcutaneous scanning, diagnosis is accurate starting on days 50-60 (left flank) or from day 90 (right flank) of pregnancy, respectively. Ultrasonography is also appropriate to examine the non-gravid uterus and the ovaries. Based on 5 years of experience working with farmed New World camelids, the article describes the physiology and pathology of reproduction in domesticated New World camelids. Particular consideration is given to the ultrasonographical examination of the genital organs.

B-mode ultrasound and grey-scale analysis of the epididymis in boars, and the relationship to semen parameters.

This study investigated the epididymis of mature boars (n=10) by means of B-mode ultrasound and grey-scale analysis (GSA) for echogenicity (EG) determination using an ultrasound unit HS 1500V, a linear transducer (frequencies 7.5-9.0 MHz), and standardized unit settings. All boars had their epididymal caput, corpus and cauda scanned six times before and after semen collection, respectively, at weekly intervals. Semen was subjected to spermatological examination including volume, total and forward motility, sperm abnormalities, as well as total sperm count and concentration. The caput and corpus both had a homogeneous fine echotexture. The cauda was homogeneous too but had a marbled echotexture. Echogenicity before and after semen collection was caput > corpus > cauda, respectively (p<0.001). Echogenicity was higher before than after semen collection for all parts of the epididymis, respectively (p<0.001). Echogenicity of the caput correlated slightly positively with the total sperm count pre-collection (r=0.301; p=0.020) and with ejaculate volume pre- and post-collection (r=0.302 vs 0.306; p=0.017 vs 0.019), and slightly negatively with sperm concentration post-collection (r= -0.275; p=0.034). No relationship was found for EG of the corpus and cauda for any of the ejaculate parameters. In conclusion, B-mode ultrasound and GSA proved feasible for imaging the epididymis in boars. Single relationships between EG and ejaculate parameters were found and deserve further investigation.

Studies on fixed-time ovulation induction in the pig.

A technology that allows for manipulating of oestrus and ovulation, and would then also allow for fixed-time insemination, can be of great benefit for swine farms that operate using sow batch management due, at least in part, to savings in labour and the production of large batches of evenly developed pigs. Thanks to the current knowledge on endocrine regulation of follicle development and ovulation, and the availability of numerous reproductively active substances such a technology is now available. It covers procedures for synchronising oestrus based on the use of altrenogest in gilts and of batch-wise weaning in sows, for stimulating follicle development using eCG and for inducing of ovulation using hCG or LH as well as GnRH analogues. While the procedures for oestrus synchronisation stand alone, other procedures require additional treatments. If fixed-time insemination is the goal, oestrus needs to be synchronised and follicular development and ovulation induced by the use of GnRH analogues and hCG with ovulation occurring within 36-42 hrs. It is a general recommendation to inseminate those animals twice, i.e. 24 and 40 hrs after ovulation induction. However, the aforementioned technology requires healthy animals and a solid management and cannot be used to compensate for poor management.

Postinsemination treatment of primiparous and multiparous cows with cloprostenol failed to affect ovulation and pregnancy rate in dairy cattle.

Previous studies have demonstrated the facilitating effect of a single cloprostenol treatment postinsemination on ovulation and pregnancy rate in dairy cattle and Italian buffalos. This study was conducted to test the effects of 500 microg cloprostenol given intramuscularly immediately postinsemination to 55 primiparous and 60 multiparous Holstein cows (Bos taurus; TR) of a typical dairy operation in East Germany and to compare them with 57 primiparous and 48 multiparous saline-treated cows (CON). Animals of the TR and CON groups did not differ or only differed marginally for age at treatment, interval calving-treatment, lactation number, milk production on the day of treatment, body condition score, and their peri- and postparturient case histories. All animals were clinically and reproductively healthy on the day of treatment. They were inseminated once 12h after the onset of estrus, scanned 24h after insemination to confirm ovulation, and tested for pregnancy by transrectal palpation between Days 42 and 48 postinsemination. Treatment did not affect the number of cows ovulating. The overall combined pregnancy rate (PR) was 47.3%. Pregnancy rate did not differ statistically between TR and CON (46.1% vs. 48.6%; P>0.05). In conclusion, this study failed to demonstrate beneficial effects of a postinsemination treatment of primiparous and multiparous cows with cloprostenol on ovulation and PR in a typical dairy cattle operation in East Germany.

Histological changes of the feline cervix, endometrium and placenta after mid-gestational termination of pregnancy with aglepristone.

This study was conducted to investigate endometrial and placental structural changes that occurred in response to mid-gestational termination of pregnancy in queens using aglepristone, a progesterone receptor antagonist. Thirteen European Shorthair pregnant queens were either treated with aglepristone (10 mg/kg body weight; subcutaneously) twice on days 25 and 26 after first mating (am; group I; n = 9), or remained untreated and served as control (group II; n = 4). Queens of group I were ovariohysterectomized between days 30 and 41 am, either at the onset (n = 3) or during (n = 1) abortion and 12 h (n = 1), 24 h (n = 3) or 10 days after abortion (n = 1). Queens of group II were ovariohysterectomized on day 30 am. Tissue was collected from the cervix, and the interplacental zone as well as the paraplacenta/placental girdle of the uterus, subjected to standard histological procedures and evaluated using light microscopy. During abortion, gaps appeared within the paraplacenta and the placental girdle which were filled with blood, leading to an embryo-maternal disconnection. Blood was also observed within the uterine lumen as well as the interstitial mucosal stroma of the cervix and the placental girdle zone and probably originated from damaged venules, whilst arterioles remained intact. As the interval between abortion and surgery increased, the interstitial and luminal haemorrhages became less pronounced and completely disappeared except interstitial remnants 10 days after abortion. The endometrial regeneration was not fully completed on day 10 after abortion and a few cystically dilated glands were evident. In conclusion, abortion of queens through aglepristone given during mid-gestation is assumed to be the result of damage of uterine venules. This leads to an interstitial haemorrhages and bleeding into the uterine lumen, subsequently resulting in utero-placental detachment.

Prevalence of PCV2 in Austrian and German boars and semen used for artificial insemination.

Since epidemiologically-based science on PCV2 in porcine semen is patchy, we investigated 806 Austrian (A) and German (G) AI boars from five studs, and boars from Austrian farms used for on-farm semen collection, for the presence for IgG/IgM in blood by ELISA (n=754) as well as for PCV2 DNA in semen (n=472) and if positive, also in blood of a few boars by nested PCR and sequencing. A total of 420 boars were tested for both PCV2 in semen and antibodies in blood. Boars were aged between 8 and 82 months at sampling. None of the boars tested positive for IgM but 60.1% did for IgG. PCV2 DNA was detected in 86 (18.2%) semen samples. Minor differences were found between boar populations with respect to the number of antibody positive boars and no differences for DNA in semen. Phylogenetic analysis of 28 sequences revealed a genetic diversity of PCV2 in semen within and between boar populations, with sequences belonging to both PCV2 genotypes 1 and 2. Mean nucleotide sequence identity was 95.7%, with maximum pairwise difference of 8.8%. Boars < or =16 months were tested more frequently positive for IgG (P<0.001) and for PCV2 DNA in semen (P<0.05) than older boars. Of 80 boars tested positive in semen, 34 (42.5%) were antibody negative. A total of 58 semen positive boars with (n=33) and without (n=25) IgG were all tested negative for PCV2 DNA in serum. In conclusion, this study demonstrated the ubiquity of PCV2 in the Austrian and German boar population. Genetically diverse PCV2 can be encountered in boar semen. Shedder boars cannot be detected on the basis of serology. There is an apparent possibility of PCV2 being transmitted through semen.

Effects of feeding level during lactation on FSH and LH secretion patterns, and follicular development in primiparous sows.

In the primiparous lactating sow undernutrition affects LH, but the effect on FSH is less clear. This study was conducted to investigate the effect of ad libitum (AL; n = 5) and restricted (RE; 70% of the AL feed; n = 5) feeding on the secretion patterns of FSH and LH, and on follicular development in primiparous lactating sows. Body side fat thickness (BSFT) was measured prior to farrowing and at weaning on day 21 postpartum (pp). Sows had an intravenous catheter fitted on day 6 or 7 pp. Blood samples were taken on days 12, 15 and 18 pp, every 15 min starting at 0700 h for 11 h, and plasma analysed for FSH and LH by radioimmunoassay (RIA). Daily transcutaneous ultrasonography of the ovaries was performed between days 10 and 20 pp to monitor follicular growth. Reduction in BSFT was higher in RE than in AL sows (p < 0.05). Mean and basal LH were significantly higher in AL than in RE sows on days 12 and 15 pp. (p < 0.05), and LH pulse frequency tended to be higher (p < 0.1). Mean FSH was higher in AL than in RE sows on days 15 and 18 (p < 0.05), and had a tendency to be higher on day 12 (p = 0.1). Follicle size increased in AL sows between days 10 and 20 of lactation (p < 0.05; r = 0.71), but remained unaffected in RE sows. In conclusion, the study demonstrates that feed restriction of primiparous sows during lactation affects not only LH, but also FSH, as well as lactational follicular growth. An association between those events is suggested.

An update on the use of B-mode ultrasonography in female pig reproduction.

After technological advances allowed for the adaptation of B-mode ultrasonography equipment for use in pig facilities, ultrasonography quickly established itself as an ideal diagnostic aid for determining pregnancy status in pigs. In recent years, B-mode ultrasonography has found increased application in its use for monitoring ovarian activity and in estimating time of ovulation in pigs. B-mode ultrasonography is also valuable in providing a detailed assessment of the sow's ovaries and uterus to determine if pathological conditions exist, which could be contributing to poor individual or herd reproductive performance. In its most recent application in pigs, the gilt genital tract has been characterized peripubertally by ultrasonography in order to detect onset of puberty. The purpose of this review is to provide an update on the current status of B-mode ultrasonography in pig reproduction, and how this technology can be of value when used in pig production medicine.

Lamprey GnRH-III stimulates FSH secretion in barrows.

Although studies have indicated that follicle-stimulating hormone (FSH) and luteinizing hormone (LH) release can be dissociated in the pig, the underlying mechanisms are still to be answered. Since it was demonstrated that lamprey gonadotropin-releasing hormone (l-GnRH-III) has preferential FSH-releasing potency in several mammalian species, we have investigated the gonadotropin-releasing activity of l-GnRH-III in barrows. Each of nine barrows (body weight: 85-90 kg; age: 207 days) received 2 ml saline (S-barrow), followed by 150 microg l-GnRH-III (1.6-1.7 microg/kg body weight) dissolved in 2 ml saline intramuscularly 7 days later. Three pre-treatment and 13 post-treatment blood samples were taken at intervals of 30 min to 8 h to assess basal and treatment-associated concentrations of FSH and LH, respectively, by radioimmunoassay. Animals were defined as having responded to treatment if, 2 h post-treatment, plasma FSH and/or LH levels were >3 SD of the respective basal concentrations. There was no treatment-associated FSH response after saline treatment, but a clear FSH response in all l-GnRH-III-injected barrows. On average, the maximum FSH level (205% of the basal concentration) was observed at 1 h post-treatment. Mean FSH values were elevated until 10 h post-treatment. There was no LH response either to saline or to l-GnRH-III. In conclusion, this study demonstrates a selective FSH-releasing activity of 150 microg l-GnRH-III in barrows. Further studies are needed to investigate whether this effect is ubiquitous in the pig and what the physiological relevance is.

Canine relaxin-like factor: unique molecular structure and differential expression within reproductive tissues of the dog.

Employing postpubertal testicular tissue, we determined the cDNA coding sequence of a truncated canine relaxin-like factor (RLF) consisting of a signal peptide of 28 amino acids (aa), a B-domain of 23 aa, a truncated C-domain of 34 aa, and an A domain of 26 aa, respectively. Within the B-domain of canine RLF, the putative relaxin receptor binding motif contained a single substitution with the C-terminal arginine replaced by a serine residue, and the putative RLF receptor binding motif was truncated. Leydig cells specifically expressed RLF in the normal postpubertal and cryptochid testis as well as in testicular Leydig cell adenoma. The epididymis was an additional source of RLF in the dog. In the female reproductive tract, expression of immunoreactive RLF and relaxin were compared. Within the ovary, RLF, but not relaxin, was detected in follicular theca interna and granulosa cells and the corpus luteum. In the nonpregnant uterus, luminal and glandular epithelium coexpressed RLF and relaxin. Uteroplacental tissue at early stages of gestation revealed RLF expression in the proliferative fetal villous cytotrophoblast and in maternal uterine cells. In the mature canine placenta, the trophoblast surrounding the maternal blood vessels and the hemophagous cytotrophoblast of the paraplacental zone expressed RLF. Canine relaxin was absent in the paraplacental areas. Western analysis of placental tissue extracts revealed the presence of specific immunoreactive bands likely resembling unprocessed and enzymatically cleaved RLF. Differential expression of RLF and relaxin appears to reflect distinct autocrine and paracrine functions of RLF in canine reproductive tissues.

Relaxin-like factor (RLF) mRNA expression in the fallow deer.

Employing RT- and RACE-PCR on RNA isolated from testicular tissue, we have cloned the coding cDNA sequence for the RLF, also known as Insl3, of the fallow deer. The RLF coding sequence consisted of 396 bp encoding a peptide of 131 amino acids and shared highest homology with bovine, sheep and goat RLF. Northern analysis revealed a single 0.9 kb transcript in the deer testis. There is only one RLF gene in the deer genome. Nonradioactive in situ hybridization revealed the Leydig cells to be the sole source for RLF mRNA in the deer testis. In the non-pregnant uterus, RLF transcripts were located in the luminal and glandular epithelium of the endometrium. Within the ovary of the pregnant doe, follicular theca interna cells and the corpus luteum expressed RLF transcripts. In uteroplacental tissues, luminal and glandular epithelium, fetal uninucleate and binucleate trophoblast cells (BNC) of the basic villous trophoblast layer expressed RLF mRNA. BNC located at the apical trophoblast layer or the tip of the fetal villus were devoid of RLF transcripts. Pseudostratified trophoblast cells at the base of fetal villi coexpressed RLF mRNA and immunoreactive MHC class Ib molecules.

Molecular cloning and localization of caprine relaxin-like factor (RLF) mRNA within the goat testis.

The relaxin-like factor (RLF) is one of the insulin-like molecules, which also includes insulin, insulin-like growth factor I and II, placentin, and relaxin. Employing RT- and RACE-PCR on RNA isolated from goat testicular tissue, we report the cloning and nucleic acid sequence of goat RLF. The caprine RLF cDNA coding sequence consisted of 396 base pairs encoding a peptide of 131 amino acids. Caprine RLF showed the highest homology in nucleic acid and amino acid sequence with bovine and sheep RLF, suggesting conservation of the RLF gene among ruminants. Northern blot analysis revealed a single 0.9 kb RLF transcript expressed in the goat testis but not in the epididymis, liver, or muscle tissue. Only a single goat RLF gene is present in the goat genome as determined by Southern blot analysis. Employing nonradioactive in situ hybridization for goat RLF mRNA and immunohistochemistry for 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17alpha-hydroxylase, we identified the Leydig cells as the sole source of RLF mRNA in the goat testis.

Canine preprorelaxin: nucleic acid sequence and localization within the canine placenta.

Employing uteroplacental tissue at Day 35 of gestation, we determined the nucleic acid sequence of canine preprorelaxin using reverse transcription- and rapid amplification of cDNA ends-polymerase chain reaction. Canine preprorelaxin cDNA consisted of 534 base pairs encoding a protein of 177 amino acids with a signal peptide of 25 amino acids (aa), a B domain of 35 aa, a C domain of 93 aa, and an A domain of 24 aa. The putative receptor binding region in the N'-terminal part of the canine relaxin B domain GRDYVR contained two substitutions from the classical motif (E-->D and L-->Y). Canine preprorelaxin shared highest homology with porcine and equine preprorelaxin. Northern analysis revealed a 1-kilobase transcript present in total RNA of canine uteroplacental tissue but not of kidney tissue. Uteroplacental tissue from two bitches each at Days 30 and 35 of gestation were studied by in situ hybridization to localize relaxin mRNA. Immunohistochemistry for relaxin, cytokeratin, vimentin, and von Willebrand factor was performed on uteroplacental tissue at Day 30 of gestation. The basal cell layer at the core of the chorionic villi was devoid of relaxin mRNA and immunoreactive relaxin or vimentin but was immunopositive for cytokeratin and identified as cytotrophoblast cells. The cell layer surrounding the chorionic villi displayed specific hybridization signals for relaxin mRNA and immunoreactivity for relaxin and cytokeratin but not for vimentin, and was identified as syncytiotrophoblast. Those areas of the chorioallantoic tissue with most intense relaxin immunoreactivity were highly vascularized as demonstrated by immunoreactive von Willebrand factor expressed on vascular endothelium. The uterine glands and nonplacental uterine areas of the canine zonary girdle placenta were devoid of relaxin mRNA and relaxin. We conclude that the syncytiotrophoblast is the source of relaxin in the canine placenta.