Low Sperm Numbers Research Articles

71 EFFECT OF CHOLESTEROL-LOADED CYCLODEXTRINS ON PRE- AND POST-THAW FUNCTION OF FELID SPERM

Propagation of genetically diverse felid populations would benefit from more effective assisted reproduction strategies, including enhanced methods for sperm cryopreservation. In felids, sperm cryopreservation has been improved by substituting soy-lecithin for egg yolk in cryomedium (Vick et al. 2012 Theriogenology 78, 2120–2128). In other species, such as elephants (Kiso et al. 2012 Reprod., Fert. Dev. 24, 1134–1142) and cattle (Purdy et al. 2004 Cryobiology 48, 36–45), the addition of cholesterol-loaded cyclodextrins (CLC) to sperm before freezing has been shown to produce superior cryopreservation results. In this study, our objectives were to (1) assess cholesterol content of cat sperm membranes and capacitation status following incubation with CLC; (2) evaluate post-thaw sperm motility, acrosome status, and fertility in vitro following CLC treatment and freezing in a soy-based cryomedium; and (3) conduct a preliminary assessment of cholesterol content in nondomestic cat sperm. Freshly collected domestic cat sperm (n = 2 males, 3–4 ejaculates/male) were incubated with CLC (0, 1.5, or 3.0 mg mL–1), and cholesterol levels were measured using an Amplex Red Cholesterol Assay. Sperm aliquots from each CLC concentration were treated with calcium ionophore (2 μM, 30 min) during in vitro incubation and stained with fluorescein isothiocyanate/PNA to evaluate induced acrosomal loss. To assess post-thaw parameters, cat sperm treated with CLC were frozen in straws using soy-lecithin cryomedium, thawed, and cultured in vitro over time. To evaluate fertility, oocytes were collected laparoscopically from gonadotropin-treated domestic cats (n = 7 females, 147 oocytes total) and inseminated with low numbers of thawed-frozen sperm pretreated with 0 or 1.5 mg mL–1 CLC. Data were analysed using ANOVA and mean differences assessed with Fisher l.s.d. or chi-squared analysis. Sperm cholesterol levels were increased (P < 0.05) after exposure to both 1.5 and 3.0 mg mL–1 CLC. Prefreeze motility was decreased (P < 0.05) and capacitation was delayed at 3.0 mg mL–1 CLC relative to treatment with 0 or 1.5 mg mL–1 CLC. Both post-thaw motility and percentage of acrosome intact sperm were reduced (P < 0.05) with the highest CLC concentration, but results were similar (P > 0.05) for 0 and 1.5 mg mL–1 CLC. Fertilization percentages did not differ (P > 0.05) between treatment groups (0 CLC, 33.3%, 25/75; 1.5 mg mL–1 CLC, 26.4%, 19/72). Preliminary results from a single cheetah (Acinonyx jubatus) and single fishing cat (Prionailurus viverrinus) suggest that sperm membrane cholesterol may be lower compared to the domestic cat. Cholesterol content appeared to increase in both species after exposure to 1.5 mg mL–1 CLC. In summary, our findings suggest CLC treatment increased cholesterol content of felid sperm membranes. The higher CLC concentration was detrimental to sperm motility, capacitation, and post-thaw sperm traits. The lower CLC concentration did not improve post-thaw sperm function in domestic cats.Research supported by the Procter & Gamble Wildlife Conservation Scholarship Program.

Current knowledge on the transport and fate of spermatozoa in the reproductive tract of the bitch.

Canine sperm transport, distribution, storage and detachment is a complex, dynamic and highly regulated process. Transport of sperm within the bitch's reproductive tract is rapid and is influenced by the method of semen deposition (natural mating or artificial insemination) and by the timing of breeding in relation to the day of ovulation. The fertile lifespan of spermatozoa in the reproductive tract of the bitch is considerably longer than in most other domestic species, and the main sperm reservoirs appear to be the uterine crypts and the distal part of the uterotubal junction, where spermatozoa attach by their heads to uterine epithelium. While several in vitro studies demonstrated prolonged motility and viability of canine spermatozoa after coincubation with uterine tube explants, spermatozoal storage has not been documented in the canine uterine tube isthmus or ampulla in vivo. Several factors, including exposure to progesterone, solubilized zona pellucida proteins and post-ovulation uterine tube fluid, appear to trigger membrane events resulting in capacitation-like changes with subsequent motility pattern changes (transitional and hyperactivated) that are associated with sperm detachment. After mating or insemination, a normal low-magnitude post-mating uterine inflammatory response occurs, evidenced by an influx of polymorphonuclear neutrophils (PMNs), increased uterine contractions and an increased uterine artery blood flow. Recently, it was also shown that normal dogs with cystic endometrial hyperplasia develop a more significant endometritis, show fewer mating-induced uterine contractions, a decreased ability of spermatozoa to bind to uterine explants in vitro and a slower uterine clearance after mating.

PP-051 - Re-freezing low numbers of sperm using cell sleepers: two case reports

PP-051 - Re-freezing low numbers of sperm using cell sleepers: two case reports

3 APPLICATION OF LAPAROSCOPIC OVIDUCTAL ARTIFICIAL INSEMINATION FOR CONSERVATION MANAGEMENT OF BRAZILIAN OCELOTS AND AMUR TIGERS

The Brazilian ocelot (Leopardus pardalis mitis) and Amur tiger (Panthera tigris altaica) are 2 iconic cat species that are becoming increasingly imperiled in the wild. Although both felids are managed in North American zoos by species survival plans (SSP), their long-term sustainability has proven difficult through captive breeding alone, necessitating the development and application of assisted reproductive techniques. Our recent progress using laparoscopic oviducal artificial insemination (LO-AI) in domestic cats (Conforti et al. 2013 Biol. Reprod. 89, 1–9) suggested that this approach could help improve reproductive management of nondomestic felids. In this study, our objectives were to (1) assess ovarian and endocrine responses to 2 exogenous gonadotropin regimens in ocelots and Amur tigers, and (2) investigate fertility and offspring production following LO-AI with freshly collected and/or frozen–thawed semen in both felid species. Female ocelots (n = 13) and Amur tigers (n = 10), housed at 16 North American zoos and recommended for breeding by the Ocelot and Tiger SSP, were treated with 2 different eCG/porcine LH (pLH) regimens (400 or 600 IU of eCG, 3000 IU of pLH, ocelots; 750 or 1000 IU of eCG, 10 000 IU of pLH, tigers). Ovarian responses were evaluated laparoscopically at 39 to 45 h post-pLH, and ovulatory females were inseminated using low numbers of freshly collected or frozen–thawed spermatozoa (≤5 million motile sperm/oviduct). Serially collected fecal samples from each female were lyophilized, extracted, and assessed via enzyme immunoassay for oestrogen and progesterone metabolite profiles. Most ocelots (11/13, 85%) and tigers (7/10, 70%) ovulated following gonadotropin treatment, with no difference (P > 0.05) between eCG dosages in mean (± s.e.m.) follicle or corpus luteum (CL) number in ocelots (11.5 ± 4.2 follicles, 2.8 ± 1.0 CL, 400 IU of eCG; 9.9 ± 5.2 follicles, 3.1 ± 1.3 CL, 600 IU of eCG) or tigers (9.0 ± 4.6 follicles, 4.0 ± 2.8 CL, 750 IU of eCG; 12.7 ± 4.4 follicles, 6.0 ± 1.7 CL, 1000 IU of eCG). Similarly, peak fecal hormone concentrations did not differ (P > 0.05) between regimens, except for slightly greater (P ≤ 0.05) progesterone levels for 10 days post-treatment with the higher eCG dose in tigers. Independent t-tests were used for all statistical calculations. One Brazilian ocelot and 1 Amur tiger conceived following LO-AI with freshly collected semen (10 × 106 motile sperm, ocelot; 0.15 × 106 motile sperm, tiger), with each producing 1 viable offspring after an 83-day and 103-day gestation, respectively. These births represent the second ocelot and first tiger produced by LO-AI. Our findings indicate that high and low eCG dosages may be equivalent and that viable offspring can be produced following LO-AI with relatively low sperm numbers in both species. Further refinement of ovarian synchronization and semen cryopreservation methods may be necessary for LO-AI to be applied routinely for ocelot and tiger conservation efforts. Funded by the Institute of Museum and Library Services, Riverbanks Zoo & Garden and Minnesota Zoo; with thanks to Ocelot & Tiger SSP, and participating zoos).

58 OVARIAN DOWN-REGULATION WITH ORAL PROGESTIN FOR FIXED-TIME LAPAROSCOPIC OVIDUCTAL ARTIFICIAL INSEMINATION WITH FRESHLY COLLECTED AND FROZEN–THAWED SPERMATOZOA IN DOMESTIC CATS

Laparoscopic oviductal AI (LO-AI) with low numbers of freshly collected or frozen-thawed spermatozoa has resulted in high pregnancy success (50–70%) in domestic cats. However, proper timing of AI depends on identifying anestrual, non-luteal queens before exogenous gonadotropin injection, confounding AI scheduling and limiting applicability with felids housed at distant institutions. Recent research (Stewart et al. 2012 Biol. Reprod. 87, 1–11) has shown that daily oral progestin treatment is effective for down-regulating ovarian activity in cats and allowing synchronized stimulation with exogenous gonadotropins. Our study objectives were to (1) assess the effect of oral progestin treatment on pregnancy success following LO-AI in domestic cats and (2) compare relative fertility following LO-AI of each female with low numbers of freshly collected versus frozen-thawed spermatozoa. Young (<2 years old), nulliparous domestic cats were assigned to either control (Con; n = 8) or oral progestin (OP; n = 7) treatment groups. Con females were monitored daily for behavioural oestrus and blood samples from anestrual females assessed for progesterone concentration to confirm non-luteal status before exogenous gonadotropin treatment [100 IU of eCG followed 85 h later with 1000 IU of porcine LH (pLH)]. Oral progestin females were fed altrenogest (Regu-Mate; 0.088 mg kg–1 of body weight) mixed in moist cat food for 38 consecutive days and then treated with exogenous gonadotropins 6 days after altrenogest cessation. At 31 to 33 h post-pLH treatment, each female was inseminated via laparoscopy in 1 oviduct with freshly collected sperm (motile) from 1 male and frozen-thawed sperm (motile; frozen in a soy lecithin-based cryomedium) from a second male. Ultrasonography was conducted approximately Day 21 post-AI for pregnancy diagnosis. Pregnant females were spayed immediately and recovered fetuses assessed for paternity using short tandem repeat molecular marker analysis to determine relative fertility of fresh versus frozen semen. All females ovulated following gonadotropin treatment, averaging ( ± standard error of the mean) 20.6 ± 1.7 corpora lutea per queen. Most [12/15 (80%)] females conceived following LO-AI, producing an average of 8.1 ± 1.4 implantations and 5.9 ± 1.2 fetuses per pregnancy. There was no difference (P > 0.05) between Con and OP cats in pregnancy success [Con: 6/8 (75%); OP: 6/7 (86%)] or in mean implantation number (Con: 6.0 ± 1.8; OP: 10.2 ± 2.0) or fetal number (Con: 4.3 ± 1.6; OP: 7.5 ± 1.6) in pregnant cats. Paternity assessment revealed that freshly collected and frozen-thawed spermatozoa were equally effective (P > 0.05) in producing pregnancies (fresh: 11/15 (73%); frozen: 10/15 (67%)], with no difference (P > 0.05) in total fetal numbers [fresh: 37/69 (54%); frozen: 32/69 (46%)]. These results indicate that oral progestin treatment may be used to down-regulate ovarian function in felids for fixed-time AI without compromising fertility in vivo, and that LO-AI with low numbers of cat sperm frozen in a soy lecithin medium may produce high pregnancy percentages and normal litter sizes.

Oligo‐astheno‐teratozoospermia in mice lacking ORP4, a sterol‐binding protein in the OSBP‐related protein family

Oligo-astheno-teratozoospermia (OAT), a condition that includes low sperm number, low sperm motility and abnormal sperm morphology, is the commonest cause of male infertility. Because genetic analysis is frequently impeded by the infertility phenotype, the genetic basis of many of OAT conditions has been hard to verify. Here, we show that deficiency of ORP4, a sterol-binding protein in the oxysterol-binding protein (OSBP)-related protein family, causes male infertility due to severe OAT in mice. In ORP4-deficient mice, spermatogonia proliferation and subsequent meiosis occurred normally, but the morphology of elongating and elongated spermatids was severely distorted, with round-shaped head, curled back head or symplast. Spermatozoa derived from ORP4-deficient mice had little or no motility and no fertilizing ability in vitro. In ORP4-deficient testis, postmeiotic spermatids underwent extensive apoptosis, leading to a severely reduced number of spermatozoa. At the ultrastructural level, nascent acrosomes appeared to normally develop in round spermatids, but acrosomes were detached from the nucleus in elongating spermatids. These results suggest that ORP4 is essential for the postmeiotic differentiation of germ cells.

Вплив препаратів з антиоксидантними властивостями на стан антиоксидантної системи сперматозоїдів при екскреторнo-токсичній формі неплідності чоловіків

Since the development of many disorders of the reproductive function in men involves processes of free radical oxidation, the purpose of this study was to form an evaluation of the pro- and antioxidant status of sperm and to restore its biological usefulness in men with excretory-toxic forms of infertility by using drugs with antioxidant properties. It is shown that excretory-toxic forms of infertility in men are mostly caused by such infectious agents as Chlamydia (22%), Chlamydia + Ureaplasma (16%), Chlamydia + Trichomonas (13%), Ureaplasma (10%). This reduces the total number of sperm in the ejaculate by 2.7 times, and motility by 1.8 times. The number of abnormal forms increases by 1.75 times. With the development of chronic inflammation of the male sex organs sperm lipid peroxidation increases by 1.3 times while the activity of glutathione peroxidase decreases (by 2.3 times) and that of glutathione reductase (by 1.7 times). We observed a close correlation between the low biological quality of sperm (low concentration, low number and motility of sperm in the ejaculate) with activation of lipid peroxidation and inhibition of activity of the glutathione antioxidant system. In the case of superoxide dismutase, the negative impact of reactive oxygen species on this enzyme was not observed. A course of drugs with antioxidant properties – vitamin E, vitamin C and zinc sulfate leads to improvement in the indicators on the spermagram (mostly sperm mobility and morphology), to reduction of the number of peroxide compounds and activation of the glutathione antioxidant system. In this case, the activity of glutathione peroxidase is increased by 1.5 times and the activity of glutathione reductase by 1.3 times. The activity of superoxide dismutase at the same time approaches the norm for zoospermia. The data obtained show that one of the pathogenic factors of the chronic inflammation of male sex organs, considered as a main developmental reason for infertility in its excretory-toxic form, is the increase in activity of the peroxide oxygen lipids of the sperm membrane and decompensation of the enzyme activity of the glutathione antioxidant system. Our data indicate that the use as medicines of vitamin E, vitamin C and zinc sulfate combined with antibiotic therapy would be highly effective in the treatment of male infertility.

Indenopyride derivative RTI-4587-073(l): A candidate for male contraception in stallions

The objective of this study was to determine whether an indenopyridine derivative RTI-4587-073(l) was a good candidate for male contraception in horses. We hypothesized that a single administration of RTI-4587-073(l) causes significant suppression of testicular function in stallions without affecting sexual behavior. Three Miniature horse stallions received a single dose of 12.5 mg/kg RTI-4587-073(l) orally (group “treated”), whereas three other Miniature horse stallions received placebo only (group “control”). Semen was collected and evaluated from all stallions twice a week for three baseline weeks and 13 post-treatment weeks. Sexual behavior was video-recorded and analyzed. Testicular dimensions were measured using ultrasonography, and blood samples were drawn for endocrine evaluation once before treatment and once a week during the post-treatment period. Single administration of RTI-4587-073(l) caused severe oligoasthenozoospermia (low sperm number and low motility), shedding large numbers of immature germ cells in semen, and increased FSH concentrations in treated stallions. These effects were fully reversible within ∼71 days. However, libido and copulatory behavior remained unchanged throughout the entire experiment. We concluded that RTI-4587-073(l) was a promising candidate for male contraceptive in domestic stallions. Further research should be performed to test this compound for fertility control in wildlife and humans.

Laparoscopic Oviductal Artificial Insemination Improves Pregnancy Success in Exogenous Gonadotropin-Treated Domestic Cats as a Model for Endangered Felids1

Artificial insemination (AI) in cats traditionally uses equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) to induce follicular development and ovulation, with subsequent bilateral laparoscopic intrauterine insemination. However, long-acting hCG generates undesirable secondary ovulations in cats. Uterine AI also requires relatively high numbers of spermatozoa for fertilization (~8 × 10(6) sperm), and unfortunately, sperm recovery from felids is frequently poor. Using short-acting porcine luteinizing hormone (pLH) instead of hCG, and using the oviduct as the site of sperm deposition, could improve fertilization success while requiring fewer spermatozoa. Our objectives were to compare pregnancy and fertilization success between 1) uterine and oviductal inseminations and 2) eCG/hCG and eCG/pLH regimens in domestic cats. Sixteen females received either eCG (100 IU)/hCG (75 IU) or eCG (100 IU)/pLH (1000 IU). All females ovulated and were inseminated in one uterine horn and the contralateral oviduct using fresh semen (1 × 10(6) motile sperm/site) from a different male for each site. Pregnant females (11/16; 69%) were spayed approximately 20 days post-AI, and fetal paternity was genetically determined. The number of corpora lutea (CL) at AI was similar between hormone regimens, but hCG increased the number of CL at 20 days post-AI. Numbers of pregnancies and normal fetuses were similar between regimens. Implantation abnormalities were observed in the hCG group only. Finally, oviductal AI produced more fetuses than uterine AI. In summary, laparoscopic oviductal AI with low sperm numbers in eCG/hCG- or eCG/pLH-treated females resulted in high pregnancy and fertilization percentages in domestic cats. Our subsequent successes with oviductal AI in eCG/pLH-treated nondomestic felids to produce healthy offspring supports cross-species applicability.

NHE8 plays important roles in male fertility

Testes have the dual function of producing sperm and testosterone. Multiple sodium/hydrogen exchanger (NHE) isoforms are expressed in the testes. NHE2 and NHE3 are expressed on the apical membrane of epithelial cells in the efferent and epididymal ducts. The testes of mice lacking NHE3 function have increased fluid volume, which leads to diminished sperm concentration and subfertility. NHE10 is an NHE isoform found in the spermatozoa. Mice lacking of NHE10 are completely infertile due to severely diminished sperm motility. NHE8 is the newest member of the NHE family. Male mice lacking NHE8 are infertile. The overall histological structure in the testes is similar between NHE8KO mice and wild type mice. NHE8KO mice have smaller testis and severely reduced serum testosterone level. NHE8KO male mice have very low number of spermatozoa in the epididymis and virtually none reached the vas deferens. PCR and Western blotting revealed that NHE8 expression is highly abundant in the testes. Immunohistochemistry located NHE8 protein in Leydig cells and in epididymal duct cells. High expression of NHE8 in the Leydig cells and low serum testosterone level in NHE8KO males strongly suggest a critical role for this new NHE isoform in Leydig cell physiology. This study is supported by NIH grant R01DK073638.

Importance of the assessment of coenzyme Q10, alpha-tocopherol and oxidative stress for the diagnosis and therapy of infertility in men

Male infertility is one of the most stressful factors of couples, being present in about 40% cases. It is usually caused by a low number of sperm (oligozoospermia) or poor sperm motility (asthenozoospermia). The sperm motility is used as an indicator of semen quality and male infertility. To the impairment of male reproduction health can contribute genetic, nutritional and environmental factors, smoking and drugs. It is well documented that excessive reactive oxygen species (ROS) production decreases sperm motility, impairs sperm function, damages the morphology of spermatozoa (1, 2). To the decreased sperm motility contribute also disturbances of sperm mitochondrial function and energy production, low levels of coenzyme Q10 and carnitine, as well as sperm mitochondrial deoxyribonucleic acid (DNA) defects. The origin of sperm dysfunction, however, is not well understood. Oxidative stress has been established as a major factor in the pathogenesis of male infertility. Low level of coenzyme Q10 contributes to the decreased sperm motility, which plays a vital role in sperm mitochondrial energy production and neutralization of reactive oxygen species (ROS).The aim of the present study was to find out, if an assessment of coenzyme Q10-TOTAL (CoQ10-TOTAL), α-tocopherol, γ-tocopherol and oxidative stress could contribute to the diagnosis of infertility in men. Two groups of infertile men, according to sperm motility (a+b and b+c) were included in the study. CoQ10-TOTAL, α-tocopherol, γ-tocopherol in plasma and seminal fluid, and parameter of oxidative stress (thiobarbituric acid reactive substances - TBARS) in plasma were determined. Higher sperm density and decreased sperm pathology were found in group a+b vs b+c (class a and b - fast and weak forward motility, class c - nonprogressive motility). Concentrations of CoQ10-TOTAL and α-tocopherol were significantly increased in seminal fluid of groups a+b vs b+c, opposite results were estimated in plasma. Concentrations of γ-tocopherol in plasma and seminal fluid of both groups were similar. Plasmatic TBARS concentrations were increased in both groups of infertile men. We suppose that incorporation of oxidative stress assessment, CoQ10-TOTAL and α-tocopherol concentrations in seminal fluid and plasma into routine andrology can play an important role for the diagnosis and targeted therapy of male infertility (Tab. 1, Ref. 16).

Laparoscopic Oviductal Embryo Transfer and Artificial Insemination in Felids – Challenges, Strategies and Successes

Embryo transfer (ET) and artificial insemination (AI) are potentially invaluable techniques for the propagation and management of genetically valuable domestic cat and endangered nondomestic cat populations. Many of the challenges that impair the effective application of ET and AI in felids may be overcome by using laparoscopic oviductal (LO) approaches. LO-ET and LO-AI are minimally-invasive procedures, requiring only two small skin incisions for insertion of a laparoscope and grasping forceps into the abdominal cavity to permit visualization and catheterization of the oviduct for embryo or semen deposition. With concurrent improvements in embryo culture systems and ovarian synchronization protocols, LO-ET has proven effective over the past decade for propagation of laboratory cats, cat models of hereditary disease and nondomestic cats. To date, viable offspring have been produced following LO-ET of non-frozen and frozen-thawed IVF-derived embryos in eight cat hereditary disease models and two nondomestic cat species, the ocelot and sand cat. LO-AI with low sperm numbers (c. 2-8 million motile) has shown similar efficacy to LO-ET, resulting in high pregnancy percentages (50-70%) following insemination of gonadotropin-treated domestic cats. Multiple kittens also have been produced in two hereditary disease models following LO-AI with frozen semen, and both ocelot and Pallas' cat kittens have been born after LO-AI with freshly-collected semen. The application of LO-ET and LO-AI to felids has resulted in substantial improvement in the efficiency of assisted reproduction for genetic management of these invaluable domestic cat and wild cat populations.

Comparative Fertility of Freshly Collected vs Frozen–Thawed Semen with Laparoscopic Oviductal Artificial Insemination in Domestic Cats

Artificial insemination (AI) is potentially invaluable as an adjunct to natural breeding for the conservation management of non-domestic felid populations. The efficacy of AI, however, must be substantially improved for applied use, especially when using frozen semen. Our recent advances in using laparoscopic oviductal AI (LO-AI) with low sperm numbers and freezing of cat semen in a soy lecithin-based cryoprotectant medium suggest that combining these two approaches might improve pregnancy outcomes with frozen-thawed spermatozoa. In this study, our objectives were to (i) assess the effect of two gonadotropin dosages (100 vs 150 IU eCG) on ovarian response in domestic cats and (ii) compare the relative fertility of frozen-thawed and fresh semen in vivo following LO-AI. All 16 females ovulated after gonadotropin treatment and were inseminated with fresh semen from one male and frozen-thawed semen from a second male. There were no differences between gonadotropin dosages in CL number, pregnancy percentage or litter size. Half (8/16) of the females conceived, with seven females giving birth to a total of 36 offspring. Paternity analysis showed that more kittens resulted from LO-AI with fresh (28/36, 78%) than frozen-thawed (8/36, 22%) semen, possibly due to impaired motility and longevity of thawed sperm. These results demonstrated that viable offspring can be produced by AI using semen frozen in a soy lecithin-based medium. Insemination with greater numbers of frozen-thawed spermatozoa, combined with further refinement of cat sperm cryopreservation methods, may be necessary to optimize pregnancy success with LO-AI in domestic and nondomestic cats.

Motile sperm organelle morphology evaluation-selected globozoospermic human sperm with an acrosomal bud exhibits novel patterns and higher levels of phospholipase C zeta

Does motile sperm organelle morphology examination (MSOME) affect levels and localization patterns of the oocyte activation factor phospholipase C zeta (PLCζ) in globozoospermic sperm with and without an acrosomal bud? MSOME identified round-headed globozoospermic sperm with increased levels of PLCζ relative to sperm from the same sample that did not undergo MSOME, and identified novel patterns of PLCζ localization in sperm exhibiting an acrosomal bud. Absence or reduction in the level of PLCζ in the sperm head, abnormal localization patterning, or defective functional ability as a result of PLCζ gene mutation, have been linked to certain types of human male factor infertility in which oocyte activation is deficient. It has been determined that a subpopulation of sperm (1%) from a patient exhibiting 100% globozoospermia presented with an acrosome bud upon MSOME. A cycle of intracytoplasmic morphologically selected sperm injection, carried out with sperm exhibiting an acrosomal bud led to pregnancy and birth of a healthy baby boy, without the use of assisted oocyte activation (AOA). Immunofluorescent analysis of PLCζ in globozoospermic sperm from three patients, before and after MSOME. Quantitative immunofluorescence was used to investigate PLCζ levels and localization patterns in individual sperm (n = 1 patient) identified by MSOME and isolated by micromanipulation, and presenting with and without the acrosomal bud. A secondary aim was to investigate levels and localization patterns of PLCζ in sperm before and after MSOME from two other globozoospermic men. Non-globozoospermic control sperm exhibited characteristic localization patterns of PLCζ immunofluorescence. Completely round-headed globozoospermic sperm from patients 1-3 were either devoid of PLCζ immunofluorescence, or exhibited an abnormal, punctate, pattern of PLCζ localization. PLCζ immunofluorescence in sperm exhibiting an acrosomal bud was observed in the midpiece with varying fluorescent intensity and was detected in 28.5% of such sperm. The majority of sperm with an acrosomal bud (43.0%) exhibited punctate patterns of PLCζ localization within the sperm head. A further 28.5% of sperm exhibited PLCζ in both the head and the midpiece. Total levels of PLCζ, and the proportions of sperm exhibiting PLCζ immunoreactivity, showed significant variance (P ≤ 0.05) amongst control [45.8 arbitrary units (a.u.) and 95.7%, respectively], non-MSOME-selected (25.9 a.u. and 46.1%, respectively) and MSOME-selected globozoospermic sperm (33.4 a.u. and 65.0%, respectively). Total levels of PLCζ immunofluorescence, and proportions of sperm exhibiting PLCζ immunoreactivity, in control sperm was significantly higher (P≤ 0.05) compared with non-MSOME-selected sperm, but not significantly different from MSOME-selected sperm. The low numbers of sperm analysed may not be ideal for conclusive statistical analysis. Evaluation of the effects of MSOME on morphologically normal sperm would confirm conclusions. The present findings provide hope for the future treatment of globozoospermia without the need for AOA, and provide further evidence for the clinical application of PLCζ as a therapeutic and prognostic tool. The research described herein was funded by the Nuffield Department of Obstetrics and Gynaecology, University of Oxford. The authors report no conflict of interest.

Microsatellite genotyping of cryopreserved spermatozoa for the improvement of whitefish semen cryobanking

The cryobanking of semen is recognized as an emerging tool for the conservation of fish biodiversity. Microsatellite analysis of the DNA of cryopreserved sperm would facilitate the assessment of genetic variability of cryobanked semen specimens. The aim of this study was to compare microsatellite profiles of DNA extracted from adipose fins and cryopreserved semen collected from eleven male whitefish (Coregonus lavaretus L.). The following microsatellite loci were employed: Cocl–Lav-8, Cocl–Lav-18, Cocl–Lav-28, Cocl–Lav-80, Str-73 and Sfo-292. The chelex 100 method was used for the successful isolation of DNA from somatic tissue, and the DNeasy method with additional modifications was used for the successful isolation of DNA from sperm. Genotyping was possible with the use of a very low number of spermatozoa (5×106 which is less than 0.1% of spermatozoa in standard 250μL straw). The results of the DNA analysis from both the adipose tissue and spermatozoa were identical. Therefore, microsatellite analysis of cryopreserved spermatozoa can be recommended for future whitefish sperm banking.

Comparison between deep intracornual artificial insemination (dIAI) and conventional artificial insemination (AI) using low concentration of spermatozoa in beef cattle

This study aimed to compare the pregnancy rate using the conventional artificial insemination (AI) or deep intracornual artificial insemination (DIAI), with low number of spermatozoa (4.0 million sperm) in 270 Nelore cows. The animals were divided in two groups (G: G1 (135 cows) conventional AI was performed (=semen deposition in the uterine body) and in G2 (135 cows) to DIAI, in ipsilateral horn where the dominant follicle in the ovary had previously been detected, by ultrasound examinations. For both the methods, a single artificial insemination was carried out after visual estrus observation, checked three times a day (morning, afternoon and evening). The pregnancy diagnosis after 45 days was conducted by ultrasound. Results showed a better pregnancy rate in the DIAI group (67.4% - p<0.01), when compared to conventional AI (48.8%) with low spermatozoa concentration.

Oncofertility and the Male Cancer Patient

Oncofertility as a discipline plays an important, adjunctive role in the treatment of male patients with cancer. Despite recommendations by the American Society of Clinical Oncology, many clinicians managing malignancies in males fail to consistently incorporate fertility preservation as a routine aspect of health care. Providers involved in the treatment of oncologic patients should have an awareness of the impact of their prescribed treatments on reproductive potential, just as they would be knowledgeable of the potential deleterious effects of cancer therapies on vital organs such as the kidneys, lungs, and liver. Providers should then have a discussion with their patients regarding these potential adverse therapeutic effects or consult a fertility preservation specialist to discuss these matters and fertility preservation options with the patient. Cryopreservation of sperm remains an excellent option for male fertility preservation as it is readily available and results in storage of viable gametes for future use in the event of post treatment infertility. With the use of assisted reproductive techniques (ART), cryopreserved sperm may ultimately result in successful paternity, even in the setting of very low numbers of stored sperm. While sperm cryopreservation is usually an option for adolescent and adult males, fertility preservation in pre-pubertal males presents a more challenging problem. To date, no clinically proven methods are available to preserve fertility in these males. However, some centers do offer experimental protocols under the oversight of an IRB, such as testicular tissue cryopreservation in these males. The hope is that one day science will provide a mechanism for immature germ cells from the testicular tissue of these patients to be used in vivo or in vitro to facilitate reproduction. In closing, studies have shown that the patient's regard for his provider is enhanced when the issue fertility preservation is raised. While oncologic care is often fraught with time constraints and acute medical concerns, fertility preservation care in the male can typically be administered quickly and without disruption of the overall plan of care.

Development and evaluation of electroejaculation techniques in the Tasmanian devil (Sarcophilus harrisii)

Electroejaculation (EEJ) has been used successfully to collect samples suitable for genome resource banking from a variety of endangered wildlife species. Ejaculates can also be used to evaluate the reproductive potential of individuals and provide information on seminal characteristics to aid in the development of sperm cryopreservation techniques. Electroejaculation techniques used for marsupial and eutherian species were tested on Tasmanian devils (n=35). Spermic ejaculates were collected in 54% (19/35) of EEJ attempts. Spermic ejaculates were low in volume (3.9±6.5×10(2) µL, range 10-3000 µL) and contained low numbers of spermatozoa (3.3±7.8×10(3) spermatozoa per ejaculate, range 6-33000). The osmolality and pH of presumptive urine-free ejaculates were 389±130 mOsm kg(-1) (range 102-566) and 7.0±0.9 (range 6.0-8.0), respectively. Prostatic bodies were observed in 79% (26/33) of ejaculates. Episodic fluctuations in serum testosterone concentrations were not detected during the EEJ procedure (P>0.05). Increases observed in serum cortisol concentrations during EEJ were less (P<0.05) than those observed after an adrenalcorticotropic hormone challenge and diurnal variation suggested that cortisol concentrations are greater during the day than at night (P<0.05). This information can be used to provide range values for the future examination of basic endocrine responses and the adrenal-pituitary axis of this species. This study also demonstrated that spermatozoa-rich devil electroejaculates are more difficult to obtain and poorer in quality than those of other marsupials.

Dicer Is Required for Haploid Male Germ Cell Differentiation in Mice

BackgroundThe RNase III endonuclease Dicer is an important regulator of gene expression that processes microRNAs (miRNAs) and small interfering RNAs (siRNAs). The best-characterized function of miRNAs is gene repression at the post-transcriptional level through the pairing with mRNAs of protein-encoding genes. Small RNAs can also act at the transcriptional level by controlling the epigenetic status of chromatin. Dicer and other mediators of small RNA pathways are present in mouse male germ cells, and several miRNAs and endogenous siRNAs are expressed in the testis, suggesting that Dicer-dependent small RNAs are involved in the control of the precisely timed and highly organised process of spermatogenesis.Principal FindingsBeing interested in the Dicer-mediated functions during spermatogenesis, we have analysed here a male germ cell-specific Dicer1 knockout mouse model, in which the deletion of Dicer1 takes place during early postnatal development in spermatogonia. We found that Dicer1 knockout testes were reduced in size and spermatogenesis within the seminiferous tubules was disrupted. Dicer1 knockout epididymides contained very low number of mature sperm with pronounced morphological abnormalities. Spermatogonial differentiation appeared unaffected. However, the number of haploid cells was decreased in knockout testes, and an increased number of apoptotic spermatocytes was observed. The most prominent defects were found during late haploid differentiation, and Dicer was demonstrated to be critical for the normal organization of chromatin and nuclear shaping of elongating spermatids.Conclusions/SignificanceWe demonstrate that Dicer and Dicer-dependent small RNAs are imperative regulators of haploid spermatid differentiation and essential for male fertility.

Therapeutic Effect of Frequent Injections of GnRH Analogue in a Beagle with Knobbed Acrosome Abnormality of Sperm

A Beagle dog (3 years old) that ejaculated high percentages (mean ± SE: 29.1 ± 1.2%) of sperm with a knobbed acrosome abnormality and a low number of sperm and that also had a low plasma testosterone (T) level was given 10 subcutaneous injections of 1 µg of gonadotropin-releasing hormone analogue (GnRH-A) at 3-day intervals. The plasma T level and number of sperm increased 12-14 weeks after the first injection. Although the percentages of sperm with knobbed acrosome abnormality did not change after the GnRH-A therapy, the number of sperm and percentage of actively motile sperm increased after the therapy, and a bitch gave birth to 5 healthy puppies after intravaginal artificial insemination with fresh semen collected 14 weeks after the first injection.