Method Of Sperm Collection Research Articles

Best practices for cryopreserving sperm in Nonhuman Primates: a systematic review and meta-analysis

Nonhuman primates (NHPs) are in increasing demand as models for preclinical research. The availability of a highly efficient cryopreservation protocol for use with NHP sperm has potential to significantly alleviate this demand and contribute to rapid increases in overall number of NHPs available for biomedical research. Thus, this systematic review assesses the state-of-the-art in NHP sperm cryopreservation and uses meta-analyses to describe the impact of varied cryopreservation approaches on NHP sperm. We searched the literature deposited in PubMed, Scopus, and Web of Science databases through June 2024 to identify data relevant to the effect of sperm cryopreservation on quality of thawed sperm compared to fresh sperm across NHP species. All original NHP studies reporting sperm parameters both before cryopreservation and after thawing were included for analysis. In total, 32 articles were included for qualitative analysis and not surprisingly, all 32 studies demonstrated negative effects of cryopreservation on sperm parameters, Specifically, motility and viability were significantly decreased in thawed NHP sperm. While the method of sperm collection did not have a significant impact on recovery of motile sperm, significant variability was evident between species. Importantly, recovery of motile NHP sperm was greatest following rapid sperm freezing methods. This analysis provides critical insights into future research directions to enhance and optimize cryopreservation protocols in NHPs.

A simple method for repeated in vivo sperm collection from laboratory mice

PurposeMouse spermatozoa for archiving laboratory mice or for in vitro fertilization (IVF) are routinely obtained from the cauda epididymis of adult males sacrificed for this purpose. To avoid the death of the donor, we tested whether a precisely timed interruption of the mating act could be used for repeated sperm collection from laboratory mice.MethodsSperm donors (B6D2F1) were mated with a receptive female, and mating behavior was observed. The stud was separated from the female 1–2 s after the onset of the ejaculatory shudder. The ejected copulatory plug with the yellowish viscous ejaculate was carefully removed from the penile cup.ResultsA total of 80 ejaculates were successfully obtained from 100 ejaculations. The latency to first mount was 1.1 ± 1.1 min (mean ± SD) and to ejaculation 8.1 ± 4.7 min. The average number of mounts to ejaculation was 10.5 ± 5.8, and the mean number of spermatozoa per collected ejaculate was 1.86 ± 1.05 × 106. An average fertilization rate of 76% was observed after IVF.ConclusionsSeparating the stud from the female just before ejaculation is feasible, easy to learn, and requires no special equipment. The sperm count of collected ejaculates is lower than natural ejaculations, but higher than previous in vivo sperm collection methods achieved. We recommend this simple sperm collection method in mice, especially when the donor cannot be sacrificed and/or repeated sperm collection from the same animal is required for experimental purposes.

Is it possible to store spotted wolffish (Anarhichas minor) sperm by refrigeration?

Spotted wolffish Anarhichas minor reproduction in captivity is dependent on in vitro fertilization. However, it is often challenging to acquire sufficient fresh sperm to fertilize the eggs that are obtained. In this study, we evaluate the possibility to store spotted wolffish sperm by refrigeration. Spotted wolffish sperm has the particularity that is already motile on stripping, and currently it is not possible to immobilize and reactivate. Thus, sperm refrigeration protocols should focus in extending this motility period that usually lasts up to 2 days. In a first experiment, we evaluated the possibility that the motility period of the sperm was limited by contamination with urine. The urea concentration in the sperm obtained both by stripping (17.10 ± 1.98 mg/dL) and directly from the testis (12.59 ± 2.37 mg/dL) was similar (p > 0.05), which indicate that the sperm collection method used avoid contamination with urine. Afterwards, we tested the possibility that the sperm motility period was limited by energy stores. The ATP concentration (initial value 5.65 ± 0.86 nmol/109 cells) remained stable (p = 0.099) during 30 h after sperm collection, and similar values (p = 0.329) were recorded at end of sperm storage in both diluted (3.88 ± 1.35 nmol/109 cells) and undiluted samples (4.76 ± 1.08 nmol/109). This indicates that the low intracellular ATP consumption, derived from the slow sperm motility, can probably be compensated rapidly enough by mitochondrial synthesis of ATP in the spotted wolffish sperm. In both experiments, diluted sperm kept higher percentage of motile cells during the storage time.

METHOD OF SEMEN COLLECTION AND ARTIFICIAL INSEMINATION IN SNAKES

This study focuses on the method for sperm extraction and artificial insemination in snakes. Ten adult healthy snakes (4.6) were included in the study (Pantherophis guttatus 1.3; Hydrodynastes gigas 1.1; Corallus hortulanus 1.1; and Sanzinia madagascariesis 1.1). Massage of the ventral aspect of the caudal third of the male snake body for 2 to 3 minutes was performed successfully for the semen collection. Both female and male P. guttatus and H. gigas were placed in brumation, with the female snakes being assessed for ovarian activity after emerging from dormancy. Only females showing ultrasonographic evidence of vitellogenic follicles were included in the study. S. madagascariensis and C. hortulanus were maintained at the same temperature through the year, and the ovarian activity was assessed ultrasonographically prior to artificially inseminating the animal. With the aid of a rigid endoscope, fresh semen was delivered through the cloaca and into the females’ oviducts using a catheter connected to the syringe. The technique failed in female S. madagascariensis and H. gigas. Two female P. guttatus laid eggs 2 months after artificial insemination, with hatchlings emerging following 2 months of development within the eggs. The third female corn snake did not lay an egg. The female C. hortulans produced eggs 4 months after insemination. The manual massage method of sperm collection in male snakes and endoscopy-assisted insemination in female snakes may be useful for conservation programs.

Characterization of pikeperch (Sander lucioperca) milt collected with a syringe and a catheter

Collection of fish milt by stripping risks contamination with urine, which may negatively influence milt characteristics and quality. Urine contamination of milt may be avoided by using a catheter for sperm collection. The objective of this study was to compare basic pikeperch milt parameters, i.e., sperm concentration (SC), pH, osmolality of seminal plasma (OSM), and motility parameters of spermatozoa analyzed with a CASA system (Computer Assisted Sperm Analysis), collected either with a syringe (as the standard method of sperm collection) or with a catheter. Every parameter of pikeperch milt collected with a catheter was significantly higher (P<0.05; SC=12.31±3.81×109ml−1, pH=9.04±0.07, OSM=311±16mOsmkg−1) than that collected with a syringe (SC=8.05±3.04×109ml−1, pH=8.93±0.09, OSM=228±51mOsmkg−1), which clearly suggests urine contamination of milt collected with the standard procedure. Moreover, milt collected with catheter was characterized by higher spermatozoa motility parameters, e.g., motility rate of sperm collected with a catheter was 73% whereas the motility rate of sperm collected with a syringe did not exceed 35%. The results obtained in this work indicate that the milt collected with the standard procedure is likely to be exposed to contamination with urine, which may cause a significant reduction of quality parameters. Collection of sperm with a catheter was proven to effectively reduce the contamination of sperm with urine. Therefore, it is recommended to collect pikeperch sperm using a catheter.

Differential distribution of sperm subpopulations and incidence of pleiomorphisms in ejaculates of captive howling monkeys (Alouatta caraya)

The aim of this study was to develop an objective method to determine the incidence of pleiomorphisms and its influence on the distribution of sperm morphometric subpopulations in ejaculates of howling monkeys (Alouatta caraya) by using a combination of computerized analysis system (ASMA) and principal component analysis (PCA) methods. Ejaculates were collected by electroejaculation methods on a regular basis from five individuals maintained under identical captive environmental, nutritional, and management conditions. Each sperm head was measured for dimensional parameters (Area [A, (square micrometers)], Perimeter [P, (micrometers)], Length [L, (micrometers)], and Width [W, (micrometers)]) and shape-derived parameters (Ellipticity [(L/W)], Elongation [(L - W)/(L + W)], and Rugosity [(4лA/P (2))]). PCA revealed two principal components explaining more than the 96 % of the variance. Clustering methods and discriminant analyzes were performed and seven separate subpopulations were identified. There were differences (P < 0.001) in the distribution of the seven subpopulations as well as in the incidence of abnormal pleiomorphisms (58.6 %, 49.8 %, 35.1 %, 66.4 %, and 55.1 %, P < 0.05) among the five donors tested. Our results indicated that differences among individuals related to the incidence of pleiomorphisms, and sperm subpopulational structure was not related to the captivity conditions or the sperm collection method, since all individuals were studied under identical conditions. In conclusion, the combination of ASMA and PCA is a useful clinical diagnostic resource for detecting deficiencies in sperm morphology and sperm subpopulations in A. caraya ejaculates that could be used in ex situ conservation programs of threatened species in Alouatta genus or even other endangered neotropical primate species.

Testicular and spermatozoan parameters in the pukeko ( Porphyrio porphyrio melanotus)

The pukeko ( Porphyrio porphyrio melanotus) is widespread in New Zealand, and is the closest living relative to the endangered takahe ( Porphyrio mantelli), which has a relatively high rate of infertility. In this study, sperm collected from a number of pukeko was analysed in order to model the reproductive physiology of the male takahe. In addition, testicular parameters were measured. To ascertain the best method of sperm collection five techniques for harvesting sperm were used on two occasions during the breeding season. All five techniques resulted in the successful recovery of sperm. However, the float-out technique produced the best quality samples. Following collection, the morphometry of unstained sperm was assessed. Our findings suggest that pukeko sperm is non-motile in the male reproductive tract. We found the mean sperm head length in the pukeko is 16.9 μm, but sperm head length varied significantly between birds. Testicular weight and length was significantly correlated with bird weight ( P < 0.05). Within each bird, testes weights were asymmetric. However, testes length was significantly correlated ( P < 0.05). There was a significant difference ( P < 0.05) in testes length between birds. The methodologies presented for obtaining and analysing pukeko sperm morphometry can be used to assist opportunistic studies of the reproductive biology of other New Zealand native birds.

113 EFFECT OF SPERM COATING ON THE QUALITY OF BOVINE FROZEN - THAWED SPERMATOZOA

The substantial decrease of sperm quality after cryopreservation remains an important issue in the artificial insemination industry. Sperm coating with Triladyl® (Minitübe, Tiefenbach, Germany) during ejaculation can preserve sperm characteristics and oocyte penetrating capacity of fresh bovine spermatozoa stored in egg yolk diluent for up to 6 days (De Pauw et al. 2003 Theriogenology 59, 1109–1122). Since collecting semen in a tube containing egg yolk-Tris extender (sperm coating) limits the period of contact between spermatozoa and seminal plasma, the present experiment was conducted to assess if this slightly adjusted method of sperm collection could also have a significant effect on bovine sperm quality after cryopreservation. Semen of five young Holstein Friesian bulls was collected by means of an artificial vagina connected to an empty tube (Group 1; five ejaculates per bull) or a tube containing 4 mL of an egg yolk-Tris extender (Groups 2 and 3; each five ejaculates per bull). The semen samples of Group 1 were conventionally diluted in straws (60 × 106 sperm/mL), frozen, and stored in liquid nitrogen. The samples of Group 3 were centrifuged, and after removing diluent and seminal plasma, the sperm pellet was conventionally diluted and processed. The samples of Group 2 were processed without removal of the supernatant. After thawing each ejaculate was analyzed for average path velocity (VAP), beat cross frequency (BCF), and progressive motility (PROG) using CASA (Minitübe, Tiefenbach, Germany). Furthermore, the membrane integrity of each sample was evaluated using fluorescent SYBR®–14/PI staining (BD Biosciences, Erembodegem, Belgium). All parameters were compared among the three groups of sperm using univariate analysis of variance (SPSS 12.0; SPSS, Inc., Chicago, IL, USA). No significant differences could be observed among the three groups for all of the evaluated sperm characteristics (Table 1). A significant effect of the bull could be determined for all analyzed parameters (P ≤ 0.02), except for the percentage of moribund cells. Nevertheless, the group-bull interaction was never statistically significant. Coating bovine sperm with an egg yolk-Tris extender during ejaculation cannot prevent the substantial deterioration of the spermatozoa that occurs during freezing and thawing since this method of sperm collection does not significantly influence the motility parameters or the membrane integrity after thawing. Table 1. VAP, BCF, PROG, and percentage of membrane-intact, dead, and moribund spermatozoa for the three groups of sperm This research was supported by IWT (no. IWT/020727).

Clinical outcomes of IVF/ICSI cycles performed with sperm obtained via epididymal aspiration vs. testicular biopsy

To determine whether there are differences in IVF outcome in patients with sperm obtained via testicular biopsy versus epididymal aspiration. Retrospective data analysis from a large private IVF center. The study included data from 98 women undergoing IVF during 2000–2002. Patients were grouped according to method of sperm collection for their IVF cycles. Thirty-six cycles utilized sperm obtained via epididymal aspiration (EA) and 62 cycles utilized sperm obtained via testicular biopsy (TB). All patients were downregulated with a standard long leuprolide acetate protocol, and underwent controlled ovarian stimulation (COH) using recombinant FSH (rFSH). Final follicular maturation was achieved with urinary hCG when the lead follicle reached at least 17 mm in diameter. Embryos were transferred on day 3. Demographic and cycle data were analyzed via the Mann-Whitney Rank Sum test. Implantation and pregnancy rates were analyzed via the chi-square test. Data are presented as medians (25%-75% range). There were no significant differences noted between the two groups in terms of demographic, stimulation or laboratory data. The EA group had a significantly higher (p=0.005) implantation rate (IR) when compared to the TB group. Clinical pregnancy and live birth rates are reported per cycle start. There was also a trend (p=0.060) towards higher clinical pregnancy rate in the EA group. Live birth rate also tended to be higher in the EA group. All data are summarized below: The group of patients with sperm obtained via epididymal aspiration had a higher implantation rate compared to the testicular biopsy group. The higher IR and trend towards higher clinical pregnancy and birth rate did not appear to be explained by differences in stimulation, follicular or oocyte data. Whether or not these differences were due primarily to variations in sperm quality between obstructive versus non-obstructive azoospermia, or due to other unrelated causes remains to be determined. Unfortunately, detailed embryo data was not available for this analysis and we are currently assessing embryo quality. Patients with EA as the source of their sperm trended towards higher IVF success than patients with TB as their sperm source, though this trend did not reach statistical significance.

Non-invasive collection of ejaculates from the common marmoset (Callithrix jacchus) using penile vibrostimulation.

Penile vibrostimulation (PVS), a noninvasive repeatable method, has been shown in the squirrel monkey to yield semen of higher quality than rectal probe electro-ejaculation (RPE). The present study aimed at establishing the conditions for PVS to collect ejaculates from marmoset monkeys. Ten adult males were trained on the appropriate handling before each was subject to six to 12 PVS tests. Ejaculation was stimulated using a FertiCare personal vibrator fitted with a 2 cm x 0.5 cm i.d. glass tube. The stimulus was repeatedly applied over a frequency of 75-95 Hz and amplitude of 1-2 mm for up to 20 min. Ejaculates were analyzed for volume, total sperm number, sperm concentration, and proportion of living and motile sperm. Ejaculates were obtained in 31 of 88 PVS tests; 87.1% of the ejaculations occurred at 80-85 Hz frequency and 1-1.5 mm amplitude. In 18 tests ejaculates were produced within 49.7 seconds. Ejaculates were characterized by (mean values): volume 31.9 microl, total sperm number 34.2 x 10(6)/ejaculate, concentration 1,154.2 x 10(6) sperm/ml, live sperm 74.6%, motile sperm 59.6%. Total number and concentration of spermatozoa were significantly enhanced in singly living males. PVS yielded three to four times more spermatozoa than comparable previously published values for RPE. Enhancing the success rate by preselecting males for responsiveness may render PVS the sperm collection method of choice in marmoset monkeys.

Spatial and temporal variation in results of purple urchin (Strongylocentrotus purpuratus) toxicity tests with zinc

AbstractPurple urchins (Strongylocentrotus purpuratus) were maintained in year‐round spawning condition in the laboratory for use in fertilization and larval development experiments designed to assess temporal variability in response to zinc. Results of these tests were compared to those from tests using gametes obtained from a field‐collected population. Fertilization and larval development tests were also conducted comparing field‐collected purple urchins from three geographically distinct groups on the West Coast of the United States. Fertilization tests conducted to assess temporal variability produced variable median effects concentrations (EC50s) ranging from 4.1 to &gt;100 μg/L zinc. Larval development tests produced more consistent EC50s, which averaged 107.4 ± 21.9 μg/L zinc for laboratory purple urchins and 97.2 ± 19.2 μg/L zinc for field‐collected purple urchins. Larval development tests did not demonstrate significant differences in response to zinc between geographically distinct purple urchin populations. Fertilization test variability was examined in terms of sperm concentration and sperm collection method during two seasons. Reduced variability was found with dry sperm collection in tests conducted in March 1995 but increased again in tests conducted in June 1995, regardless of sperm collection method. Increased variability in response to zinc may be caused by seasonal temperature effects.

Bulk sperm collection by epididymal micropuncture and stimulation of the spermatic nerve; a novel method for sperm retrieval for IVF for surgically irreparable vasal obstruction.

We have developed a new sperm collection method by epididymal micropuncture in conjunction with perivascular nerve stimulation to obtain as many uncontaminated spermatozoa as possible for IVF for patients with surgically unreconstructable vasal obstruction. Fifteen azoospermic men with congenital absence of the vas deferens or vasal obstruction not amenable to surgical repair enrolled in this study. Under general anaesthesia the testis and epididymis were retrieved and stabilized in a testicular holder with the aid of 2% agar. The epididymal micropuncture was performed using a micropipette placed in a micromanipulator. A pair of Ag-AgCl ring electrodes were placed around the spermatic cord for perivascular nerve stimulation with DC (intensity 136 V, frequency 20 Hz) for 30 s. Electrical stimulation was repeated every 1 min until no further fluid was obtained. Epididymal fluid obtained ranged from 78 to 980 microliters (mean 169 microliters). Sperm concentration ranged from 0.7 to 200 million (mean 53.5 million). Sperm motility ranged from 0.5% to 70% (mean: 19.9%). The percentage of spermatozoa with normal morphology ranged from 3% to 32% (mean 16.4%). Of 16 cycles in which spermatozoa and oocytes were retrieved, 56% (9/16) resulted in fertilization and 18.8% (3/16) developed clinical pregnancies. The clinical pregnancy rate per couple was 20% (3/15). This is the first clinical experience of the bulk sperm collection system for IVF for the treatment of unreconstructable obstructive azoospermia. Our results indicate that our new technique, in conjunction with IVF, is clinically useful for patients with surgically irreparable vasal obstruction.

A new sperm collection method for in-vitro fertilization: collection at tail of epididymis.

We have developed a method for the collection of spermatozoa from the tail of the epididymis in patients with severe asthenozoospermia. The treatment was applied to 25 cycles, of which 16 cycles provided spermatozoa in good condition with concentration and motility greater than 50 x 10(4)/ml and greater than 50% respectively. Spermatozoa thus obtained were subjected to IVF in 11 cycles and to ZIFT in five cycles; five IVF cases reached cleavage and two of those cases resulted in pregnancy. Two of the five ZIFT cases also achieved a pregnancy. Despite limits on frequency, the sperm collection process is thought to be very useful for the treatment of severe asthenozoospermia.

The method of sperm collection significantly influences sperm motion parameters following ethane dimethanesulphonate administration in the rat

Sperm motion analysis following exposure to a reproductive toxicant is one means of evaluating the functional integrity of the testis and epididymis. In this study we sought to determine whether the method used to collect sperm from the proximal cauda epididymidis, where sperm are not completely mature, has a significant influence on sperm motion parameters. Two methods of collecting rat sperm for motion analysis were used: one based on an aspiration technique selected from the literature; the other, a new approach based on diffusion of sperm from the epididymal tubule. The two methods were tested for sensitivity to effects on sperm motility parameters 4 days after a single exposure to ethane dimethanesulphonate (EDS). Since EDS is known to decrease serum testosterone (T), an additional group of rats received T-filled implants just prior to dosing to determine if the decrease in serum T alone had an effect on sperm motility. The results of the study yielded strikingly different interpretations of the effect of a 65 mg/kg BW dose of EDS on the motility of sperm taken from the proximal cauda epididymidis. Sperm collected by “aspiration” showed no significant decrease in the percentage of motile or progressively motile sperm compared to vehicle-treated animals. On the contrary, sperm collected by “diffusion” showed large, significant decreases in the percentages of both motile and progressively motile sperm. This difference was due largely to lower percentages of motile and progressively motile sperm in control sperm samples collected by aspiration. Similarly, the motion parameters of sperm collected by the aspiration method were unaffected by EDS/T treatment. By contrast, sperm collected by the diffusion method showed complete restoration of the percentages of motile and progressively motile sperm, and progressive velocity was significantly, but incompletely, restored by T implantation. Statistical analyses indicate that the diffusion method of sperm collection provides enhanced sensitivity, as well as precision of the data, thereby decreasing the number of animals required in a treatment group to detect a statistically significant alteration of a sperm motion parameter.