Seminal lipid profiling and antioxidant capacity: A species comparison.

On their way to the oocyte, sperm cells are subjected to oxidative stress, which may trigger the oxidation of phospholipids (PL). Applying MALDI-TOF MS, HPTLC and ESI-IT MS, we comparatively analyzed the PL compositions of semen and blood of species differing in their reproductive systems and types of nutrition (bull, boar, stallion, lion and man) with regard to the sensitivity to oxidation as well as the accumulation of harmful lyso-PL (LPL), transient products of lipid oxidation. In addition, the protective capacity of seminal fluid (SF) was also examined. The PL composition of erythrocytes and blood plasma is similar across the species, while pronounced differences exist for sperm and SF. Since the blood function is largely conserved across mammalian species, but the reproductive systems may vary in many aspects, the obtained results suggest that the PL composition is not determined by the type of nutrition, but by the relatedness of species and by functional requirements of cell membranes such as fluidity. Sperm motion and fertilization of oocytes require a rather flexible membrane, which is accomplished by significant moieties of unsaturated fatty acyl residues in sperm lipids of most species, but implies a higher risk of oxidation. Due to a high content of plasmalogens (alkenyl ether lipids), bull sperm are most susceptible to oxidation. Our data indicate that bull sperm possess the most effective protective power in SF. Obviously, a co-evolution of PL composition and protective mechanisms has occurred in semen and is related to the reproductive characteristics. Although the protective capacity in human SF seems well developed, we recorded the most pronounced individual contaminations with LPL in human semen. Probably, massive oxidative challenges related to lifestyle factors interfere with natural conditions.

Effects of Different Freezing Protocols on Motility, Viability, Mitochondrial Membrane Potential, Intracellular Calcium Level, and DNA Integrity of Cryopreserved Equine Epididymal Sperm.

The aim of the present study was to evaluate the effect of different freezing procedures on sperm motion, viability, the acrosome status, mitochondrial membrane potential (MMP), intracellular calcium content, and DNA integrity on epididymal stallion sperm. Therefore, the sperm of 10 healthy stallions was harvested by retrograde flushing after testectomy, diluted with a semen extender containing defined milk proteins and a freezing extender containing egg yolk and glycerol and frozen according to 4 different protocols, using a programmable freezer and a floating rack performing a slow (processes 1 and 2) or a fast cooling rate (processes 3 and 4, respectively). Post-thaw total motility and slow sperm values were lower when using process 4 compared with processes 1 and 2 (P < .05) after 1 hour of incubation. Progressive motility was lower in process 4 compared with process 1 immediately after thawing and after 1 hour of incubation (P < .05). The amount of rapid sperm was lower when using process 4 compared with process 1 immediately after thawing (P < .05). After 1 hour of incubation, the amount of rapid sperm was lower when using process 4 compared with processes 1 and 2 (P < .05). Higher values for viable sperm were seen in processes 1 and 2 compared with process 4 (P < .05) after 1 hour of incubation. Immediately after thawing, more viable sperm with high MMP (hMMP) were observed when using process 3 compared with process 2 (P < .05). After 1 hour of incubation, a significantly higher amount of viable hMMP sperm were detected when using processes 1 and 2 compared with process 4 (P < .05). Process 2 yielded a lower percentage of sperm containing low calcium (lCa) than process 3 immediately after thawing (P < .05). After 1 hour of incubation, the lowest amount of lCa sperm was observed using process 4 (P < .05). The subpopulation of viable/hMMP/lCa sperm was higher when using process 3 compared with process 2 immediately after thawing (P < .05). After 1 hour of incubation, the lowest amount of this subpopulation was detected in process 4 (P < .05). The DNA integrity was similar in all groups. In conclusion, a slow cooling rate with a controlled rate freezer resulted in best sperm quality after thawing. Using a floating rack in nitrogen vapor as an alternative to a programmable freezer, equilibration in a cooled environment is advantageous.

Comparison of the Effects of Five Semen Extenders on the Quality of Frozen-Thawed Equine Epididymal Sperm.

Cryopreservation of epididymal sperm allows the saving of genetic material in case of unexpected death or emergency castration. The aim of the present study was the comparison of five different combinations of extenders commercially available for equine frozen semen processing for cryopreservation of epididymal sperm. Epididymal sperm were harvested from gonads of 10 healthy stallions after routine castration by retrograde flush technique. Then, samples were split and diluted with (1) INRA96 + INRA Freeze, (2) BotuSemen + BotuCRIO, (3) EquiPlus + Gent Freeze, (4) EquiPlus + EquiPlus Freeze, and (5) Gent + Gent Freeze. Extenders 1 and 2 showed higher values for total and progressive motility after thawing compared with extender 4 (P < .05). Extender 3 was in between 1 and 2 (P > .05), and extender 5 resulted in the lowest values (P < .05). The subpopulation of viable frozen-thawed sperm with high mitochondrial membrane potential and low intracellular calcium content was higher using extender 1 compared with extenders 3, 4, and 5 (P < .05) and higher in extender 2 compared with extenders 4 and 5 immediately after thawing (P < .05). After 1 hour of incubation, this subpopulation yielded the highest values in extender 2 (P < .05). Immediately after thawing, extender 1 yielded higher values for percentage of DFI and mean DFI than extenders 3, 4, and 5 (P < .05). Following 1 hour of incubation after thawing, sperm processed with extender 1 resulted in the highest values for percentage of DFI and mean DFI (P < .05). Using extender 2, mean DFI values were lower than those in extender 1 and higher than the extenders 3, 4, and 5 (P < .05). The study revealed that according to the examined sperm quality parameters, freezing extenders (extender 1, extender 2) using low concentrations of glycerol either combined with or without methylformamide were beneficial for cryopreservation of stallion epididymal sperm. For processing of stallion epididymal sperm, an extender containing milk proteins (extenders 1-4) for initial dilution after sperm harvesting is preferable to an extender including egg yolk (extender 5).

[Birth of a foal following insemination with frozen-thawed epididymal sperm and simultaneous intrauterine delivery of homologous seminal plasma]. / Geburt eines Fohlens nach Besamung mit tiefgefrorenen Nebenhodenspermien und zeitgleicher intrauteriner Verabreichung von homologem Seminalplasma.

Cryopreservation of epididymal sperm allows final preservation of the gene reserve from valuable sires in case of unexpected injury terminating the breeding career. This case report describes the birth of a healthy foal following insemination with frozen-thawed epididymal sperm. The testes and epididymides were removed under general anaesthesia and sent cooled to the laboratory overnight. The cauda epididymidis was dissected and 17.79 × 109 sperm were harvested by a retrograde flush technique. A fertile mare was inseminated 1 year later with frozen-thawed epididymal sperm. Sperm were deposited into the tip of the uterine horn ipsilateral to the ovulation site. The mare did not become pregnant after the 1st cycle. In the 2nd breeding cycle, additional homologous seminal plasma was delivered into the uterus at the time of insemination and the mare was diagnosed as pregnant 14 days post ovulation. A healthy colt was born after 334 days of gestation. The method for preparing gonads for transportation to an appropriate laboratory is described for veterinarians and the different steps of semen collection and preservation are presented.

Fertility and 63,X Mosaicism in a Haflinger Sibship.

Chromosomal abnormalities are notable causes of infertility in horses. Mares show various degrees of estrous behavior, and ultrasound examination often reveals an underdeveloped genital tract. This article reports investigations on fertility in a Haflinger sibship with a healthy, normally developed, fertile mare with at least three healthy offspring. Chromosomal analysis performed incidentally and blinded for this mare revealed 63,X/64,XX/65,XXX mosaicism. Two closely related mares were also mosaics (63,X/64,XX), and one of them was a carrier of a marker chromosome. Repeated examinations of the mare and seven relatives (four mares and three stallions) did not provide evidence for sub- or in-fertility. They had no developmental abnormalities or conspicuous body conditions. Peripheral blood samples were collected for analysis of the karyotype and molecular analyses. Chromosomes were Giemsa stained and 4',6-diamidino-2-phenylindole banded to identify numerical or structural aberrations of chromosomes and identification of sex chromosomes, respectively. Fluorescence in situ hybridization was performed with an equine Y-chromosome painting probe to identify and count the sex chromosomes, and polymerase chain reaction analysis was used to test for the presence of the SRY gene and investigating chimerism. The present article demonstrates the necessity of further studies analyzing chromosomal X0 mosaics to improve the predictive value of chromosomal aberrations on fertility.

Postthaw Addition of Autologous Seminal Plasma Improves Sperm Motion Characteristics in Fair and Poor Freezer Stallions.

During semen processing for cryopreservation, most seminal plasma is usually removed, and components with protective effects on sperm may be missing after thawing and within the female reproductive tract. The present study evaluated the effect of postthaw addition of autologous seminal plasma on motion characteristics of stallion sperm with fair (n = 4) or poor (n = 3) freezability. Therefore, pure seminal plasma (group SP1), seminal plasma combined with fresh semen extender (group SP2), or seminal plasma mixed with freezing extender (group SP3) were used to fill 0.5 mL straws and frozen similar to stallion semen. Postthawing, semen samples (n = 42) were diluted either with semen extender (group FT) or with seminal plasma (n = 126) of groups SP1 to SP3 to 25 × 106 sperm/mL. In fair freezer stallions, total and progressive motilities were higher in group FT than in group SP1 (P < .05), but there was no difference in poor freezing stallions among groups (P > .05). However, comparing individual stallions, positive effects of seminal plasma on total or progressive motility were detected in two stallions. Curvilinear velocity increased in groups SP2 and SP3 in fair freezer stallions and in all groups with seminal plasma compared with group FT in poor freezer stallions (P < .05). Although straightness was higher in groups SP2 and SP3 compared with group FT in fair freezer stallions (P < .05), there was no difference among groups in stallions with poor freezability (P > .05). Average lateral head displacement did not change among groups of fair freezer stallions (P > .05) but was higher in groups SP2 and SP3 than in group FT in poor freezer stallions (P < .05). Beat cross frequency was higher in all groups diluted with seminal plasma postthawing in fair freezer stallions (P < .05), but only in group SP1 than in group FT in poor freezer stallions (P < .05). The addition of autologous seminal plasma to frozen-thawed semen can improve motion characteristics of stallions with fair and poor freezability. This is a valuable additional protocol for laboratories dealing with cryopreservation of stallion semen and for veterinarians working with fair or poor freezer stallions.

New monoclonal antibodies specific for mammalian protamines P1 and P2.

The expression of protamines and the binding of these small arginine-rich proteins to DNA complete the process of spermatid chromatin reorganization and the global inactivation of the male's haploid genome that occurs during the final stages of sperm development in mammals. While a number of anti-protamine antibodies have been created during the last 40 years, only a few have proven useful for detecting the presence of the protamines, determining the timing of their expression and deposition in chromatin, and investigating their structure and function in both maturing spermatids and sperm. The aim of this effort was to develop an additional set of monoclonal antibodies (MAbs) that not only recognize new P1 and P2 protamine epitopes but also work well as IHC reagents for detecting and identifying mammalian protamines in testicular tissue and ejaculated sperm. Using a combination of native and synthetic human protamines as antigens, 38 hybridoma clones recognizing human protamine P1 or P2 were generated. Antibodies produced by the 12 best clones were screened for selectivity by enzyme-linked immunosorbent assay, and two were found to recognize only human protamine P1 or P2, while a number of the others bound to both the human and mouse proteins. One MAb recognized every protamine tested. All the antibodies, including one recognizing stallion P1 and another recognizing stallion P2, bound to the native protamines in the chromatin of spermatids or sperm. While the majority labeled only elongating spermatids or sperm, several of the antibodies were found to also bind to the cytoplasm or nuclei of cells that lack protamine, which indicates these MAbs must recognize epitopes present in the protamines that are also found in other proteins. Thirteen overlapping human protamine P1 peptides were synthesized and subsequently used to identify the epitopes recognized by the six best antibodies. Abbreviations: BSA: bovine serum albumin; ELISA: enzyme-linked immunosorbent assay; HCl: hydrochloric acid; IHC: immunohistochemistry; i.p: intraperitoneal; LIS: lithium diiodosalicylate; MAb: monoclonal antibody; PBS: phosphate buffered saline.

Disorder of Sexual Development in a Mare with an Unusual Tentative Mosaic Karyotype: 63,X/64,Xdel(Y).

The present report describes a 4-year-old Trakehner mare which was referred to the clinic for a breeding soundness evaluation. Clinical, histological, and postmortem examination revealed an underdeveloped genital tract, the absence of a cervix uteri, and small inactive ovaries without male gonadal tissue. Blood lymphocyte analysis revealed an unusual mosaic karyotype consisting of 2 cell lines. For the majority of cells (70%), monosomy X (63,X) was observed. The remaining cells (30%) contained 64 chromosomes including one X chromosome and a small rudimentary Y chromosome consisting mostly of heterochromatin. The centromere was retained, but its full functionality was questionable. PCR analysis revealed that the entire male-specific region of Y (Yq14), including the SRY gene, was deleted. It remained unclear if the pseudoautosomal region (Yq15) and parts of the heterochromatic region (Yq13) were affected by this deletion. The phenotype of the mare with this disorder of sex development associated with sex chromosome abnormalities is genetically comparable to 63,X monosomy which fully explains the clinical findings.

[Clinical signs in late pregnant mares]. / Klinische Parameter in der Spätträchtigkeit bei der Stute.

OBJECTIVE: During the peripartal period, interpretation of basic clinical signs may be challenging. In the present study, heart rate (HR), respiratory rate (RR) and body temperature (BT) were evaluated in healthy mares of different breed types and compared to reference values for adult horses from the literature. MATERIAL AND METHODS: During daily physical exams of periparturient mares, the HR, RR and BT were evaluated. Differences according to the horse's size were investigated and in large breeds, the influence of dystocia or retained placenta was analysed. RESULTS: During the last weeks before parturition (a. p.), the HR significantly increased and was clearly lower after parturition (p. p.; p < 0.05). In larger horses, the RR increased a. p. and decreased p. p. (p < 0.05). The BT underwent changes in all groups during the periparturient period and was higher p. p. (p < 0.05). In general, values for HR, RR and BT were highest in ponies (p < 0.05) while the lowest RR was measured in large horses (p < 0.05). There was no difference in the HR between mares with eutocia or with dystocia (p > 0.05). By contrast, the RR was significantly higher in mares with dystocia on day 1 p. p. (p < 0.05). Differences in the BT a. p. and p. p. occurred only in mares with eutocia (p < 0.05) and remained within the normal values. Mares with retained placenta did not exhibit significant changes in the HR (p > 0.05), but the BT was higher on day 1 p. p. (p < 0.05). CONCLUSION: Increased HR, RR and BT in mares during late pregnancy suggest a distinct physical performance for a prolonged period of time. Interpretation of these parameters in relation to the mare's reproductive state is essential to diagnose potential disorders and to determine whether therapy is required.

Concentrations of altrenogest in plasma of mares and foals and in allantoic and amniotic fluid at parturition.

Treatment with the progestin altrenogest is widely used in pregnant mares. The fact that foals born from healthy mares treated with altrenogest until term suffered from neonatal problems raises the question of direct effects of altrenogest on vital functions in the neonate. We have therefore investigated altrenogest concentrations in maternal and neonatal blood plasma and in fetal fluids. Pregnant mares were treated with altrenogest orally once daily (0,088 mg/kg bodyweight, n = 7) or left untreated (n = 8) from 280 d of gestation until foaling. Altrenogest concentration was determined in plasma of the mares, their foals and in amniotic and allantoic fluid. The concentration of altrenogest in plasma from treated mares (2.6 +/- 1.0 ng/mL) was significantly lower than in plasma from their foals immediately after birth (5.6 +/- 1.9 ng/mL; p < 0.05), but was significantly higher than in their fetal fluids (amniotic fluid: 0.4 +/- 0.1 ng/mL; p < 0.05; allantoic fluid: 3.0 +/- 1.5 ng/mL). Altrenogest was undetectable in maternal and fetal plasma and fetal fluids of control pregnancies at all times. Altrenogest concentration in plasma of foals from treated mares was strongly correlated to the altrenogest concentration in plasma of their dams (r = 0.938, p < 0.001) and in amniotic (r = 0.886, p < 0.001) and allantoic fluid (r = 0.562, p < 0.05). A significant decrease in altrenogest concentration between the time periods 0-15 min, 30-120 min, and 180-360 min after parturition was seen in the plasma from foals born to altrenogest-treated mares. In conclusion, our data demonstrate that altrenogest reaches the equine fetus at high concentrations.