Epididymectomy as a novel surgical procedure; application in the domestic cat.

Feline overpopulation raises issues concerning health, ecology, economy, and ethics. Procedures to limit overpopulation should carefully address animal welfare, efficiency, costs, and feasibility. Vasectomy in unowned cats is suggested as preferable to standard neutering as it maintains male sexual behaviour which may induce ovulation and pseudopregnancy in intact females and may prevent immigration of other males. Vasectomy is not performed routinely because it is fastidious, time consuming and requires more material than standard neutering. We describe epididymectomy as an alternative. In a first experiment, we analysed semen, testosterone, behaviour and pain in six experimental cats before and after epididymectomy, and after castration two months later. Excised tissues were analysed histologically. Testosterone concentrations did not differ significantly between intact and epididymectomised animals but were significantly different after castration. Sexual behaviour and testicular spermatogenesis persisted after epididymectomy, but with a marked drop in the semen count after 7 days. The Glasgow pain scores did not differ significantly after epididymectomy and castration. In a subsequent experiment, 20 privately owned cats were epididymectomised and castrated immediately afterwards, to analyse the learning curve and perioperative complications. The time required for an epididymectomy was significantly shorter than for castration. The study confirms that epididymectomy is quicker and less invasive than castration, it is associated with minimal risks and post-operative pain while easy to learn and inexpensive. Further field studies are required to test its efficiency for feline feral population control or in other species such as in bears, lions or deer, where infertility is required and castration not wanted.

In Vitro Evaluation of the Permeability of Different Resorbable Xenogeneic Membranes after Collagenolytic Degradation.

In this in vitro study, we compare the penetration of cells through different resorbable collagen membranes, which were collagenolytically degraded over different time periods. Three different resorbable collagen membranes were evaluated, including two non-cross-linked (NCL) membranes-namely, a porcine (NCL-P) and an equine (NCL-E) membrane-and an enzymatically cross-linked porcine (ECL-B) membrane. A special two-chamber model was fabricated, allowing for the placement of separating membranes, and a non-porous polyester membrane was used as a negative control (C), in order to verify the impermeability of the experimental chamber device. Round membrane samples with a diameter of 16 mm were fabricated. Eighteen membranes of each type were punched and placed on polyethylene nets as carriers. The membranes were then biodegraded-each on its carrier-in 12-well polystyrene plates: three samples of each membrane type were degraded for 1.5, 3, 6, or 12 h in 2 mL of a buffered collagenase solution, at 37 °C. For control purposes, three samples of each membrane type were not degraded, but only immersed in buffer solution for 1.5, 3, 6, or 12 h, at 37 °C. Another three samples of each type of membrane were degraded until complete dissolution, in order to determine the full hydroxyproline content for comparison. Liquid-preserved boar semen (containing at least 120 million sperm cells per milliliter) was used to test the cell occlusivity of the degraded membranes. At baseline and initial degradation, all tested membranes were tight, and no penetration was observed with up to 30 min of incubation time (results not shown). After 1.5 h, cells were partially capable of penetrating the NCL-E membrane only. One sample showed leakage, with a sperm volume of 1.7 million cells/mL over all samples. No penetration occurred in the test, NCL-P, and ECL-B groups. After a degradation time of 3 h, the NCL-P and ECL-B membranes remained occlusive to cells. All the membranes and measurements indicated leakage in the NCL-E group. After 6 h, four NCL-P measurements showed the first signs of cell penetration, as boar spermatozoa were detectable in the lower chamber (64 million cells/mL). The ECL-B membranes remained completely cell occlusive. After 12 h, four NCL-P measurements were cell penetration positive (14.6 million cells/mL), while the ECL-B group remained tight and showed no cell penetration. As the findings of our study are well in accordance with the results of several previous animal studies, it can be concluded that the surrogate model is capable of performing rapid and cheap screening of cell occlusivity for different collagen membranes in a very standardized manner. In particular, claims of long degradation resistance can be easily proven and compared. As the boar spermatozoa used in the present report had a size of 9 × 5 µm, smaller bacteria are probably also able to penetrate the leaking membranes; in this regard, our proposed study set-up may provide valuable information, although it must be acknowledged that sperm cells show active mobility and do not only translocate by growth.

A 1-bp deletion in bovine QRICH2 causes low sperm count and immotile sperm with multiple morphological abnormalities.

BACKGROUND: Semen quality and insemination success are monitored in artificial insemination bulls to ensure high male fertility rates. Only ejaculates that fulfill minimum quality requirements are processed and eventually used for artificial inseminations. We examined 70,990 ejaculates from 1343 Brown Swiss bulls to identify bulls from which all ejaculates were rejected due to low semen quality. This procedure identified a bull that produced 12 ejaculates with an aberrantly small number of sperm (0.2 ± 0.2 × 109 sperm per mL) which were mostly immotile due to multiple morphological abnormalities. RESULTS: The genome of this bull was sequenced at a 12× coverage to investigate a possible genetic cause. Comparing the sequence variant genotypes of this bull with those from 397 fertile bulls revealed a 1-bp deletion in the coding sequence of the QRICH2 gene which encodes the glutamine rich 2 protein, as a compelling candidate causal variant. This 1-bp deletion causes a frameshift in translation and a premature termination codon (ENSBTAP00000018337.1:p.Cys1644AlafsTer52). The analysis of testis transcriptomes from 76 bulls showed that the transcript with the premature termination codon is subject to nonsense-mediated mRNA decay. The 1-bp deletion resides in a 675-kb haplotype that includes 181 single nucleotide polymorphisms (SNPs) from the Illumina BovineHD Bead chip. This haplotype segregates at a frequency of 5% in the Brown Swiss cattle population. Our analysis also identified another bull that carried the 1-bp deletion in the homozygous state. Semen analyses from the second bull confirmed low sperm concentration and immotile sperm with multiple morphological abnormalities that primarily affect the sperm flagellum and, to a lesser extent, the sperm head. CONCLUSIONS: A recessive loss-of-function allele of the bovine QRICH2 gene likely causes low sperm concentration and immotile sperm with multiple morphological abnormalities. Routine sperm analyses unambiguously identify homozygous bulls for this allele. A direct gene test can be implemented to monitor the frequency of the undesired allele in cattle populations.

Autosomal recessive loci contribute significantly to quantitative variation of male fertility in a dairy cattle population.

BACKGROUND: Cattle are ideally suited to investigate the genetics of male fertility. Semen from individual bulls is used for thousands of artificial inseminations for which the fertilization success is monitored. Results from the breeding soundness examination and repeated observations of semen quality complement the fertility evaluation for each bull. RESULTS: In a cohort of 3881 Brown Swiss bulls that had genotypes at 683,609 SNPs, we reveal four novel recessive QTL for male fertility on BTA1, 18, 25, and 26 using haplotype-based association testing. A QTL for bull fertility on BTA1 is also associated with sperm head shape anomalies. All other QTL are not associated with any of the semen quality traits investigated. We perform complementary fine-mapping approaches using publicly available transcriptomes as well as whole-genome sequencing data of 125 Brown Swiss bulls to reveal candidate causal variants. We show that missense or nonsense variants in SPATA16, VWA3A, ENSBTAG00000006717 and ENSBTAG00000019919 are in linkage disequilibrium with the QTL. Using whole-genome sequence data, we detect strong association (P = 4.83 × 10- 12) of a missense variant (p.Ile193Met) in SPATA16 with male fertility. However, non-coding variants exhibit stronger association at all QTL suggesting that variants in regulatory regions contribute to variation in bull fertility. CONCLUSION: Our findings in a dairy cattle population provide evidence that recessive variants may contribute substantially to quantitative variation in male fertility in mammals. Detecting causal variants that underpin variation in male fertility remains difficult because the most strongly associated variants reside in poorly annotated non-coding regions.

Infertility due to defective sperm flagella caused by an intronic deletion in DNAH17 that perturbs splicing.

Artificial insemination in pig (Sus scrofa domesticus) breeding involves the evaluation of the semen quality of breeding boars. Ejaculates that fulfill predefined quality requirements are processed, diluted and used for inseminations. Within short time, eight Swiss Large White boars producing immotile sperm that had multiple morphological abnormalities of the sperm flagella were noticed at a semen collection center. The eight boars were inbred on a common ancestor suggesting that the novel sperm flagella defect is a recessive trait. Transmission electron microscopy cross-sections revealed that the immotile sperm had disorganized flagellar axonemes. Haplotype-based association testing involving microarray-derived genotypes at 41,094 SNPs of six affected and 100 fertile boars yielded strong association (P = 4.22 × 10-15) at chromosome 12. Autozygosity mapping enabled us to pinpoint the causal mutation on a 1.11 Mb haplotype located between 3,473,632 and 4,587,759 bp. The haplotype carries an intronic 13-bp deletion (Chr12:3,556,401-3,556,414 bp) that is compatible with recessive inheritance. The 13-bp deletion excises the polypyrimidine tract upstream exon 56 of DNAH17 (XM_021066525.1: c.8510-17_8510-5del) encoding dynein axonemal heavy chain 17. Transcriptome analysis of the testis of two affected boars revealed that the loss of the polypyrimidine tract causes exon skipping which results in the in-frame loss of 89 amino acids from DNAH17. Disruption of DNAH17 impairs the assembly of the flagellar axoneme and manifests in multiple morphological abnormalities of the sperm flagella. Direct gene testing may now be implemented to monitor the defective allele in the Swiss Large White population and prevent the frequent manifestation of a sterilizing sperm tail disorder in breeding boars.

Herbal yeast product, Equi-Strath®, alters the antioxidant status of stallion semen.

Effects of a plasmolysed yeast product enriched with herbs, malt, honey and orange syrup on semen characteristics and oxidative status in stallions were evaluated. Twenty stallions (mean age ±â€¯standard deviation = 9.5 ±â€¯4.5 years) were randomly divided into a treatment group (n = 10) receiving 0.06 mL/kg bodyweight of plasmolysed herbal yeast, and a control group (n = 10) receiving the same amount of placebo daily in the feed for 10 weeks. Ejaculates were collected weekly from all stallions starting at Week 0. Volume, sperm concentration, motility, and velocity were evaluated immediately, 24 and 48 h after cooled storage at 5 °C. At the two storage time points, membrane lipid peroxidation was determined using the BODIPY-C11. Additionally, blood samples were collected at Weeks 0, 1, 5 and 9, and analysed for antioxidant status, consisting of superoxide dismutase, cholesterol, thiobarbituric acid reactive substances, and non-esterified fatty acids. Due to the nature of the data, the Mann-Whitney U test was applied as preliminary analysis. The BODIPY-C11 in the semen was less at 24 h and greater at 48 h after collections in Week 1 to 3 (P < 0.01) and Week 1 to 10 (P <  0.05) compared with Week 0 in the treatment compared to control group. There were no significant differences between groups for all values for other seminal and blood variables evaluated. In conclusion, feed supplementation with plasmolysed herbal yeast temporarily improved the antioxidant status of stallion semen, which might be of benefit for preservation of cooled semen.

Development of a flow cytometric assay to assess the bacterial count in boar semen.

The aim of the study was to develop a new flow cytometric assay for the determination of the bacterial count in commercially processed boar semen. In total 224 fresh boar semen samples collected at an AI-station were analyzed. The number of total viable counts (TVC) was determined by using flow cytometry after staining with SYBR Green I and Propidium Iodide (PI). In the first part of the study 111 fresh boar semen samples were spiked with pure cultures of defined numbers of bacteria commonly detected in boar ejaculates and analyzed by flow cytometry. In the second part, 113 fresh semen samples were assessed on the day of collection through flow cytometry and the Most Probable Number (MPN) method, as the standard bacteriological method. The first part of the study showed a strong correlation between the detected and expected numbers (r = 0.96; P < 0.001), while in the second part of the study the TVC determined by flow cytometry and by the MPN method correlated only moderately (r = 0.28; P < 0.01; median MPN: 24,000 ±â€¯MAD 21,600 bacteria/mL; median flow cytometry: 24,426 ±â€¯MAD 15,610 bacteria/mL). In summary flow cytometry is a fast alternative to the classical culture technique to determine highly contaminated boar ejaculates. The developed flow cytometric protocol enables one to enumerate the viable bacteria within fresh boar ejaculates without requiring numerous treatment steps, and thus offering the possibility of an on-line use in AI-centers.

Correction to: Insemination with border disease virus-infected semen results in seroconversion in cows but not persistent infection in fetuses.

The original article [1] contained an error whereby a co-author, Sarah Züblin had their name displayed incorrectly. This error has now been corrected.

Insemination with border disease virus-infected semen results in seroconversion in cows but not persistent infection in fetuses.

BACKGROUND: This study examined various health variables in cows after artificial insemination with Border disease virus (BDV)-infected semen and the occurrence of persistent infection in ensuing fetuses. Five cows were inseminated (day 0) with BDV-infected semen as well as with semen from a fertile Eringer bull. One cow, inseminated with virus-free semen only, served as a control. Clinical examination, assessment of eating and rumination activities, measurement of intraruminal temperature and leukocyte count were used to monitor the health of the cows. Blood samples were collected at regular intervals for the detection of viral RNA and antibodies against BDV, and the cows were slaughtered on day 56. The uteri, placentae and fetuses were examined macroscopically, histologically, immunohistochemically and by means of molecular methods for the presence of pestiviruses. RESULTS: The demeanour, eating and rumination activities and intraruminal temperature were not affected by insemination with BDV-infected semen, whereas the total leukocyte and lymphocyte counts dropped transiently and were significantly lower on day 6 than on day 0. Seroconversion occurred by day 28 in the five infected cows but not in the control cow. The uteri, placentae and fetuses had no macroscopic or histological lesions, and immunohistochemical examination and RT-PCR were negative for pestiviruses. CONCLUSIONS: The findings showed that cows inseminated with BDV-infected semen seroconverted and fetuses thus produced were not persistently infected. Transmission of BDV to cattle through infected semen, therefore, seems to be of minor importance.

Immunization against GnRF in adult cattle: a prospective field study.

BACKGROUND: Suppression of cyclic activity in cattle is often desired in alpine farming and for feedlot cattle, not intended for breeding. A cattle specific anti-GnRF vaccine (Bopriva™) is registered for use in heifers and bulls in different countries. In adult cows vaccinated with Bopriva™, the median period until recurrence of class III follicles was 78 days from the day of the 2nd vaccination and reversibility could be proven, as out of 11 experimental cows 10 cows became pregnant at first, and one cow at second insemination. In the present study, 76 healthy, cyclic Eringer heifers and cows were vaccinated twice with Bopriva™ 3-7 weeks apart, to prevent estrus during alpine pasturing. Blood samples were taken for progesterone and GnRF antibody titer analysis on the day of inclusion (7-9 d before the first vaccination) and at the first vaccination. At the same time, gynaecological examinations were performed. When estrus occurred in the course of the alpine pasturing season, a gynaecological examination was done including analysis of a blood sample (progesterone, anti-GnRF antibody titer). Cows were followed for fertility out to 26 months post second vaccination. RESULTS: Median duration of estrus suppression was 191 days after the second vaccination (when the 2 vaccinations were given 28-35 days apart). From n = 13 cows showing signs of estrus on the alpine pasture, n = 7 could not be confirmed in estrus (serum progesterone value >2 ng/ml, no class III follicles seen using ultrasonography). Median duration between second vaccination and next calving was 496 days (25%/75% quartiles: 478/532 days). CONCLUSION: Bopriva™ induced a reliable and reversible suppression of estrus for more than 3 months in over 90% of the cows.

Computed tomographic findings and treatment of a bull with pituitary gland abscess.

BACKGROUND: In cattle, the prognosis of brain abscess is unfavourable and treatment is therefore not recommended. To the knowledge of the authors, there has been no report of successful treatment of a brain abscess in cattle.This report describes the clinical, computed tomographic and postmortem findings in a Holstein-Friesian bull with a hypophyseal abscess. CASE REPORT: The main clinical findings were generalised ataxia, ptyalism, prolapse of the tongue, dropped jaw, dysphagia, head tilt and unilateral ptosis. Cerebrospinal fluid evaluation revealed 2437 leukocytes/µl and severe pleocytosis. CT examination of the head showed a cavitary lesion consistent with an abscess in the hypophysis. Treatment consisted of gentamicin and flunixin meglumine for 3 days and amoxicillin for 40 days. The neurological signs resolved within 8 days of the start of treatment. The bull was slaughtered 11 months later because of infertility, and a postmortem examination was carried out. Histologically, a mild chronic non suppurative meningoencephalitis restricted to the ventral diencephalon was diagnosed. In addition, there was mild to moderate multifocal chronic lymphoplasmacytic hypophysitis with mild multifocal fibrosis. CONCLUSIONS: This case report stresses the significance of CT in confirming the clinical and laboratory diagnosis of central nervous system disorders in cattle and for localising brain lesions. Treatment of the brain abscess resulted, with respect to the central nervous disorder, in a successful outcome and was encouraging considering that most cases have an unfavourable prognosis.

Transmission of border disease virus from a persistently infected calf to seronegative heifers in early pregnancy.

BACKGROUND: This study describes the transmission of border disease virus (BDV) from a persistently infected calf to seronegative heifers in early pregnancy, resulting in persistently infected fetuses. On day 50 of pregnancy (= day 0 of the infection phase), six heifers were co-housed in a free stall with a bull calf persistently infected with BDV (pi BVD) for 60 days. The heifers underwent daily clinical examination, and blood samples were collected regularly for detection of pestiviral RNA and anti-pestivirus antibodies. After day 60 (= day 110 of pregnancy), the heifers were slaughtered, and the fetuses and placentae underwent post-mortem and immunohistochemical examination and RT-PCR for viral RNA detection. RESULTS: Three heifers had mild viraemia from day 8 to day 14, and by day 40 all heifers had pestivirus antibodies identified as anti-BDV antibodies in the serum neutralisation test. The placenta of the three viraemic heifers had histological evidence of inflammation, and fetal organs from these heifers were positive for pestivirus antigen by immunohistochemical examination and for BD viral RNA by RT-PCR and sequencing. Thus, co-housing of heifers in early pregnancy with a pi-BDV calf led to seroconversion in all heifers and persistent fetal infection in three. CONCLUSIONS: Considering that pi-BDV cattle can infect other cattle and lead to persistent infection of the fetus in pregnant cows, BDV should not be ignored in the context of the mandatory BVDV eradication and monitoring program. This strongly suggests that BDV should be taken into account in BVD eradication and control programs.

37,X/38,XY Mosaicism in a Cryptorchid Bengal Cat with Müllerian Duct Remnants.

A 23-month-old tomcat was referred to our clinic because of male behavioral problems, cryptorchidism, and an undefined intra-abdominal organ resembling a uterus. Ultrasonography and computed tomography showed 2 fluid-filled tubular structures dorsolaterally to the bladder and connected to the pelvic urethra. The cat was castrated, and the tubular structures were surgically removed. Histology identified them as Müllerian duct remnants. The testes were hypoplastic, the epididymes and deferent ducts were normal. Cytogenetic analyses revealed the presence of a mosaic 37,X/38,XY karyotype which explains the clinical findings.

Germ cell transplantation in an azoospermic Klinefelter bull.

Germ cell transplantation is a technique that transfers donor testicular cells into recipient testes. A population of germ cells can colonize the recipient testis, initiate spermatogenesis, and produce sperm capable of fertilization. In the present study, a nonmosaic Klinefelter bull was used as a germ cell recipient. The donor cell suspension was introduced into the rete testis using ultrasound-guided puncture. A pulsatile administration of GnRH was performed to stimulate spermatogenesis. The molecular approach to detect donor cells was done by a quantitative polymerase chain reaction with allele discrimination based on a genetic mutation between donor and recipient. Therefore, a known genetic mutation, associated with coat-color phenotype, was used to calculate the ratio of donor to recipient cells in the biopsy specimens and ejaculates for 10 mo. After slaughtering, meiotic preparations were performed. The injected germ cells did not undergo spermatogenesis. Six months after germ cell transplantation, the donor cells were rejected, which indicates that the donor cells could not incorporate in the testis. The hormone stimulation showed that the testosterone-producing Leydig cells were functionally intact. Despite subfertility therapy, neither the recipient nor the donor cells underwent spermatogenesis. Therefore, nonmosaic Klinefelter bulls are not suitable as germ cell recipients. Future germ cell recipients in cattle could be mosaic Klinefelters, interspecies hybrids, bulls with Sertoli cell-only syndrome, or bulls with disrupted germ cell migration caused by RNA interference.

Analysis of the pathogenetic basis for shedding and transmission of ovine gamma herpesvirus 2.

Ovine herpesvirus 2 (OvHV-2), a member of the viral subfamily Gammaherpesvirinae, shares numerous similarities with human herpesvirus 8 (HHV-8). Both viruses are apathogenic in their healthy original host, may cause lymphoprolipherative diseases, cannot routinely be propagated in cell culture, and may be sexually transmitted. However, the pathways of sexual transmission of these viruses, as well as the underlying pathogenetic dynamics, are not well understood. Organs from naturally OvHV-2-infected, as well as OvHV-2-free, sheep were quantitatively analyzed for OvHV-2 by the DNA amplification techniques. The dynamics of OvHV-2 multiplication and excretion were monitored after experimental infections and, most importantly, subsequent to vasectomy. The OvHV-2 DNA load in various tissues and internal organs was not merely reflecting the viral DNA load in the bloodstream, which suggested compartmentalization of OvHV-2. Moreover, OvHV-2 DNA was detected at several portals for virus shedding, i.e., the respiratory, alimentary, and urogenital tracts. Transient OvHV-2 excretion was detected in ejaculates of experimentally infected rams. Upon vasectomy, OvHV-2 DNA reappeared in the ejaculatory plasma, but the titers did not decline after reaching a peak. Spiking and fractionation experiments revealed an inhibitory activity, associated with the spermatozoa, which was able to suppress detection of viral DNA but which was no longer present in samples from vasectomized animals. Therefore, epidemiological studies on viruses that may be transmitted by the ejaculatory pathway and for whose tracing nucleic acid amplification methods are used, i.e., OvHV-2, HHV-8, and the human immunodeficiency virus, should include vasectomized males.

Absence of the prion protein homologue Doppel causes male sterility.

The agent that causes prion diseases is thought to be identical with PrP(Sc), a conformer of the normal prion protein PrP(C). PrP(C)-deficient mice do not exhibit major pathologies, perhaps because they express a protein termed Dpl, which shares significant biochemical and structural homology with PrP(C). To investigate the physiological function of Dpl, we generated mice harbouring a homozygous disruption of the Prnd gene that encodes Dpl. Dpl deficiency did not interfere with embryonic and postnatal development, but resulted in male sterility. Dpl protein was expressed at late stages of spermiogenesis, and spermatids of Dpl mutants were reduced in numbers, immobile, malformed and unable to fertilize oocytes in vitro. Mechanical dissection of the zona pellucida partially restored in vitro fertilization. We conclude that Dpl regulates male fertility by controlling several aspects of male gametogenesis and sperm-egg interaction.