Effect Of Sperm Concentration Research Articles

Effects of sperm concentration at semen collection and storage period of frozen semen on dairy cow conception

The present study was based on data obtained from artificial inseminations (AIs) performed with cryopreserved semen from elite bulls used in the Norwegian breeding program. Semen was diluted to standardize the number of spermatozoa to 18 million per AI dose. The aim of the study was to investigate whether the net sperm concentration at semen collection and the storage period in liquid nitrogen have any effect on probability of conception in dairy cattle. We demonstrated that the natural range of sperm concentration at semen collection within some of the bulls was associated with the probability of conception. However, no primary trend among bulls was found on the effect of sperm concentration at semen collection. This appears to be due to differences among bulls in their response to the dilution ratio of seminal plasma to extender. The effect of storage time was investigated in semen that had been stored between 1000 days and 2400 days in AI straws in liquid nitrogen at the AI center. Our findings showed that use of semen with the longest storage period, i.e. 1951–2400 days, resulted in a more than one percentage point lower probability of conception than semen with a shorter storage period. In conclusion, the net sperm concentration at semen collection, which affects the dilution ratio of seminal plasma to extender, should be considered individually among bulls to achieve optimal reproductive performance. Furthermore, this study gives support to the idea that a measurable degree of damage to the spermatozoa could occur during the preservation time in liquid nitrogen.

Gamete interactions and fertilization behavior in the larvacean, Oikopleura dioica

Summary Oikopleura dioica are often cultured in the laboratory but for several reasons are clearly not in conditions similar to those of the open sea. Despite this impediment, some interesting behaviors of the spawning of adults and gametes in 3-ml well plates were observed from April and May 1992–1994 using the circulation devices of Fenaux and Cie and Gorsky and Fenaux (2002). Testis sac size measurements and observations of swimming behavior of numerous individual males were made during sperm release. We calculated the intervals between initiation of sperm release and its cessation, the sinking rates of individual eggs, the potential for re-fertilization of unfertilized eggs up to 24 h post release, and the effects of sperm concentration on fertilization success. We found that in a group of 74 males the total minutes of spawning time was 1,076 min with an average time to completion of spawning by all males at 14.54 min. Individual eggs sink in a 10-ml graduated cylinder at the rate of 2.35 mm/s in still water at 16°C. During sperm shedding, most males made a series of from one to five small horizontal loops every few seconds along the upper edge of a 3-ml well. The number of loops made increased during spawning. We suggest that this behavior is a decreasing regular spiral. Most males make clockwise loops while swimming against the inner edge of the wells but a few individuals rotated in the counterclockwise direction. Sperm aging was studied using sperm 100 min or older prior to the addition of newly spawned eggs. After fresh sperm were added to small groups of 24-h-old eggs incubated in 3-ml wells, several eggs cleaved properly and developed to the twitching larval stage. Fertilization success curves obtained on three different days using separate batches of eggs and sperm from each male produced nearly identical curves. However, sperm left standing at 16°C for longer than 100 min rapidly lost potency. Addition of higher sperm concentrations dramatically increased the fertilization rate.

Efeito de concentração espermática e período de incubação oócitoespermatozóides na fecundação in vitro em bovinos da raça Gir

Avaliou-se o efeito de concentrações espermáticas e períodos de incubação na fecundação in vitro com sêmen de touros Gir (Bos indicus). Oócitos maturados in vitro foram fecundados com sêmen de dois touros, nas concentrações de 1, 2 ou 4x10(6) espermatozóides/mL, por 12 ou 18 horas, e avaliada a taxa de fecundação e polispermia. Posteriormente, foi avaliada a taxa de clivagem com 72 horas e de blastocisto no oitavo dia pósfecundação. Um segundo experimento verificou o efeito de 1, 2 e 4x10(6) espermatozóides/mL por 18 horas, utilizando sêmen de três touros separadamente, bem como o efeito de 12 e 18 horas de incubação, na concentração de 1 a 2x10(6) espermatozóides/mL. Observou-se maior (P<0,05) polispermia na concentração de 4x10(6) espermatozóides/mL e que a associação dessa concentração com 18 horas aumentou a taxa de clivagem (P<0,05). No segundo experimento, o aumento do período de incubação melhorou (P<0,05) a taxa de clivagem para um dos touros e, quando comparados entre si, os animais produziram taxa de clivagem e de blastocistos diferentes (P<0,05) em uma mesma concentração ou período de incubação. Os resultados revelaram que a polispermia é o principal efeito do aumento da concentração espermática e que a eficiência entre touros na fecundação in vitro é dependente da concentração e período de incubação.

Development of in vitro embryo production systems for red deer ( Cervus elaphus) : Part 3. In vitro fertilisation using sheep serum as a capacitating agent and the subsequent birth of calves

The following experiments investigated the use of sheep serum (SS) as a capacitating agent for red deer ( Cervus elaphus) sperm during in vitro fertilisation. Red deer oocytes were collected at slaughter and matured in vitro for 24 h in TCM-199 supplemented with 10% foetal calf serum, 10 μg ml −1 FSH and LH, and 1 μg ml −1 of oestradiol. Fertilisation medium was IVF-SOF modified to contain 5 mM Ca 2+ and no glucose. Experiment 1 investigated the addition of heparin, BSA (8 mg ml −1) or 20% SS. All oocytes were penetrated when IVF-SOF was supplemented with SS compared to 10 and 0% penetration when either heparin or BSA was present ( P<0.01). However, 43.8% of these oocytes were polyspermic when the medium contained SS. In Experiment 2, the effect of sperm concentration on penetration rates during in vitro fertilisation was investigated. Total sperm penetration and monospermic penetration rates increased with increased sperm concentrations in a log linear manner ( P<0.001) and both approached an asymptote at 0.4×10 6 sperm ml −1 with 93.6 and 77% for total and monospermic penetration, respectively. Polyspermic fertilisation also increased with increasing sperm concentrations ( P<0.05) but was variable (range 3.5±4.2 to 42.3±10.6%), especially at the lower sperm concentrations. Experiment 3 investigated the viability of these oocytes after transfer into red deer recipients. Fifteen 2- and 4-cell embryos were transferred into the oviducts of synchronized recipients 28 h post in vitro insemination. An additional fourteen embryos (8–10 cell) were transferred into synchronised recipients after 48 h of in vitro culture in either SOFaaBSA ( n=10) or on red deer epithelial oviduct monolayers ( n=4). Five (33% 5/15) of the recipients that received 2- and 4-cell embryos were pregnant at Day 45 (verified by ultrasonography) and four recipients subsequently calved. One recipient receiving an embryo cultured in SOFaaBSA was pregnant at Day 45 and subsequently calved. The birth of five normal calves indicate that full developmental competence of red deer oocytes matured and fertilised in vitro can be achieved by the techniques described.

Fertilizability and developmental capacity of individually cultured bovine oocytes

Culture of single oocytes throughout in vitro maturation (IVM), fertilization (IVF) and culture (IVC) provides detailed information on maturity, fertilizability and developmental capacity of individual bovine oocytes and embryos. In the present study, effects of sperm concentration (Experiment 1), microdrop size (Experiment 2), and the addition of hypotaurine (HT) or glutathione (GSH; Experiment 3) during IVF were investigated. In Experiment 4, in vitro maturity and developmental capacity of bovine oocytes cultured for IVM in a medium supplemented with fetal calf serum (FCS), bovine serum albumin (BSA) or polyvinyl alcohol (PVA) during IVM were investigated. In Experiments 1 to 3, the percentages of normal (2 pronuclei with a spermtail) and polyspermic fertilization in singly cultured oocytes were similar to those of group IVF culture (5 oocytes/drop). The addition of GSH during single oocyte IVF significantly increased the proportion of normal fertilization and decreased the polyspermic fertilization compared with addition of HT or of the control. The rates of mature oocytes (62.4 and 67.7%) and blastocyst development (12.9 and 15.2%) for single oocyte IVM cultures (Experiment 4) were also similar compared with the group culture; PVA supplementation significantly increased the matured oocyte rate, but decreased blastocyst development significantly (7.1%) as compared with FCS (19.5%) or BSA (15.6%). These results indicate that a single oocyte culture system throughout in vitro production of bovine embryos provides similar maturity, fertilizability and developmental capacity to oocytes cultured in groups.

Use of a sperm analyzer for evaluating broiler breeder males. 1. Effects of altering sperm quality and quantity on the sperm motility index

A new instrument for assessing mammalian semen attributes, the Sperm Quality Analyzer, was evaluated as a potential tool for determining rooster sperm quality. The Sperm Quality Analyzer measures the “activity” of sperm in a semen sample as the sperm motility index (SMI). The SMI is defined as the number and amplitude of deflections in a light path per second as a result of sperm movement within a capillary tube. In the present study, effects of sperm concentration, viability, and motility on the SMI were evaluated. Peterson broiler breeder males (n = 40) were used as semen donors. In the initial experiment, semen was diluted from 2- to 25-fold and SMI readings were obtained. The SMI was very low in neat semen samples but increased when semen was diluted up to threefold. However, at dilutions greater than fivefold, the SMI decreased. Apparently, sperm concentration in undiluted semen is so great that sperm are unable to move freely within the capillary tube. Maximum SMI values were obtained at sperm concentrations of approximately 1 billion sperm per milliliter. When thawed, dead sperm were mixed with incubated, live sperm, the SMI decreased with decreasing sperm viability even though sperm concentration was constant. Obviously, fewer sperm move across the light beam as sperm mortality increases. When motile, aerobically incubated sperm were mixed at different rates with immotile, anaerobically incubated sperm samples, the SMI increased with increasing concentrations of motile sperm, whereas total sperm concentration was static. In addition, the SMI was strongly correlated with motility scores obtained by microscopic analysis. The Sperm Quality Analyzer provides an estimate of the overall quality of sperm from broiler breeder males by reflecting sperm concentration, viability, and motility in a single value, the SMI.

Effects of sperm concentration and egg number on fertilization efficiency with channel catfish ( [formula omitted]) eggs and blue catfish ( [formula omitted]) spermatozoa

The mean sperm concentration of 10 blue catfish ( Ictalurus furcatus ) was 1.03 × 10 10 per gram of testis. Testis weighed 3.9 and 17.2 g, with a mean of 6.6 g per fish. Fertilization rate of channel catfish ( Ictalurus punctatus ) eggs fertilized with 5.00 × 10 4 to 1.20 × 10 7 blue catfish spermatozoa per egg was 17 to 87%, with an overall mean of 65%. Sperm concentrations of 5.0 × 10 4/egg exhibited a lower, 16.6% (P < 0.05) fertilization rate than higher sperm concentrations (1.25 × 10 5 to 1.20 × 10 8/egg). Batches of 450, 2,000, 5,000, 8,000 and 11,000 eggs were similarly fertilized with various sperm concentrations. Mean fertility rate ranged from 25 to 67%, with an overall mean of 53%. The largest egg mass produced the lowest (P < 0.05) fertilization rate. A combination of 450 eggs per batch and 5.0 × 10 5 to 1.20x 8 sperm per egg produced the highest rate of fertilization (67 to 87%).

Effects of time and sperm concentration on reactive oxygen species formation in human semen.

Although generally accepted standards exist for routine semen analysis, recent methods of assessing reactive oxygen species (ROS) in human semen lack a standardized protocol. The purpose of this study was to investigate (1) the relationship between ROS level and the time interval between semen collection and analysis, and (2) the effect of sperm concentration on the level of ROS formation. Semen specimens from men (n = 40) consulting for infertility treatment were divided in two groups: in 20, routine semen analysis was performed and ROS formation evaluated at 1, 3, 5, and 24 h after semen collection; in the other 20, ROS formation was evaluated at four sperm concentrations (60, 30, 15, and 7.5 x 10(6)/mL). White blood cell (WBC) concentration was assessed before ROS measurement using a myeloperoxidase staining technique (Endtz test). ROS level was measured by a chemiluminescence method. ROS formation decreased significantly over time. The mean ROS level 343.4 (1 h), 133.5 x 10(4) cpm (3 h, p = .004), 66.0 x 10(4) cpm (5 h, p < or = .001), and 22.2 x 10(4) cpm (24 h, p < or = .001), respectively. In the first group of 20 specimens, 14 were positive for ROS formation at 1 h after collection, and 4 of these were positive for the Endtz test (> 1 x 10(6) WBC/mL). The number of ROS-positive specimens after 3, 5, and 24 h was eight, six, and two, respectively. In the second group, eight patients were positive for ROS formation at 1 h after collection.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphology and subsequent development in culture of bovine oocytes matured in vitro under various conditions of fertilization

The objective of these experiments was to evaluate factors affecting in vitro fertilization of bovine oocytes matured in vitro, and their subsequent development to blastocysts. In Expts 1 and 2, sperm concentration, spermatozoa and oocyte incubation time, motility enhancers and semen source were manipulated. Fluorescent microscopy of microtubules and chromatin was used to observe sperm penetration rate, sperm aster formation and chromatin decondensation. Oocyte penetration rates were affected by sperm concentration but not by spermatozoa and oocyte incubation time. The effect of sperm concentration was due primarily to changes in polyspermy and not monospermy. Motility enhancers had no effect on any parameter measured. In Expt 3, oocytes were matured for 17, 22, 28 and 34 h before fertilization and evaluated for fertilization rates, morphology of cortical granules and exocytosis and blastocyst development. A domain free of cortical granules that was associated with the metaphase chromatin was not observed in mature bovine oocytes. As oocytes matured from 17 to 34 h, the distribution of cortical granules progressed from clustered to diffuse. Although monospermic fertilization rates were similar and cortical granule exocytosis occurred in all groups, polyspermy increased with maturation time. Development to blastocysts increased from 17 to 22 h of maturation but decreased thereafter with increasing maturation time. These results suggest that polyspermy can be reduced by adjusting sperm concentration and spermatozoa and oocyte incubation time with little effect on monospermic fertilization. Increased polyspermy with increased maturation time was not due to a lack of cortical granule exocytosis.

Effect of sperm concentration during preincubation in a defined medium on fertilization in vitro of pig follicular oocytes.

Pig follicular oocytes cultured in a defined medium for 28-29 h were inseminated in vitro by epididymal or ejaculated boar spermatozoa that were preincubated in a modified KRB solution at various sperm concentrations for 4 h at 37 degrees C. Sperm concentration at insemination was 2 X 10(6) cells/ml. When epididymal spermatozoa were preincubated at concentrations of 4-16 X 10(8) cells/ml, 71-75% of oocytes were penetrated. In contrast, preincubation at a low concentration (0.8 X 10(8) cells/ml) resulted in a low penetration rate (11%). Epididymal spermatozoa preincubated at a concentration of 4 X 10(8) cells/ml could also penetrate denuded oocytes. None of the oocytes were penetrated by epididymal spermatozoa that were exposed to seminal plasma before preincubation or by ejaculated spermatozoa. After preincubation, whiplash motility was observed in the epididymal spermatozoa, but not in the ejaculated spermatozoa.

Effect of sperm concentration in the zona-free hamster ova penetration assay

Effect of sperm concentration in the zona-free hamster ova penetration assay

The effect of sperm concentrations during preincubation and at insemination on fertilization of rat eggs in vitro.

Rat epididymal spermatozoa, at a concentration of 40-65 X 10(4) cells/ml, were preincubated for 5-5, 5 hr and then added to eggs from superovulated, immature rats. By 10-12 hr after insemination, 86% of the eggs had undergone fertilization, but many of them (41%) were polyspermic. When sperm were preincubated at almost the same concentration and for the time stated above, but then diluted to 1-3 X 10(4) cells/ml before insemination, the number of fertilized eggs and the incidence of polyspermy were both reduced, being 56% and 5%, respectively. When a sperm concentration of 1-3 X 10(4) cells/ml was used both during preincubation and at insemination, the fertilization rate of 60% or more, with polyspermy not in excess of 10%, a sperm concentration of 30-100 X 10(4) cells/ml during preincubation and then diluted to approximately 1-3 X 10(4) cells/ml at insemination appears to be optimal.

Effects of sperm concentration on the capacitation of rat spermatozoa.

AbstractRat epididymal spermatozoa were preincubated for various times in concentrated (9.6–14.9 × 106 sperm/ml) or diluted form (0.7–1.2 × 106 sperm/ml) and mixed at similar concentrations with eggs recovered from superovulated immature rats. When the eggs were examined at various times after insemination, the penetration rates, the time of sperm penetration, and the fertilizing capacity of sperm were greatly reduced following the preincubation of spermatozoa in a concentrated form for 5–7.5 hours. By preincubation of sperm in diluted form, capacitation of spermatozoa was achieved in 3–5.5 hours, the penetration rates were increased, and the interval between insemination and sperm penetration was progressively reduced as the duration of preincubation increased up to 7.5 hours. It appears that the sperm concentration plays an important role for the capacitation of rat spermatozoa in vitro and the maintenance of their fertilizing capacity.

Effect of sperm concentration on the oxygen uptake of bull semen.

Effect of sperm concentration on the oxygen uptake of bull semen.

Relationship of Hyaluronidase Concentration to Fertility of Dairy Bull Semen

The possibility that the concentration of the enzyme hyaluronidase in mammalian semen is a critical factor in fertility has led the authors to investigate this relationship in dairy bulls. In fertility studies in rabbits, Rowlands (8) found that by adding seminal plasma from killed spermatozoa suspensions containing hyaluronidase to dilute spermatozoa suspensions the median effective spermatozoa concentration for fertility was reduced to one-sixth of that of the controls. In similar experiments, however, Chang (1) indicated that the increased fertilizing capacity obtained by adding seminal plasma to dilute suspensions of spermatozoa was not due to hyaluronidase but to some other seminal plasma factor. Seminal plasma in which the hyaluronidase had been inactivated by heat was similarly effective, whereas added bull testes hyaluronidase had no effect. Kurzrok et al. (5) reported that in six cases of human female infertility where the female was apparently not at fault and where the male seminal hyaluronidase concentration was low, the application of bull testes hyaluronidase to the uterine cervix with subsequent coitus resulted in pregnancies. Later, Kurzrok (4) reported that 33 out of 102 similar clinical patients conceived following the application of bull testes hyaluronidase to the uterine cervix. Entirely negative results were obtained by Seigler (10) in a series of 48 cases of human female infertility where hyaluronidase was applied and where the female presumably was not at fault. Semen samples from the male partner were not assayed for hyaluronidase in these cases. A zero order correlation coefficient of -0.32 (significant at the 5 per cent level) between hyaluronidase titer and fertility of dairy bull semen was obtained by Sallman and Birkeland (9). The hyaluronidase assays in this case were made within 20 hr. of the time of ejaculation. In considering the negative correlation which they obtained, these authors suggested that the removal of hyaluronidase from semen might improve the fertility of the semen. In this general connection it is interesting to note that Johnston and Mixner (3) found a first order partial correlation (limiting effect of sperm concentration) of -0.30 (significant at the 5 per cent level) between percentage of live spermatozoa in dairy bull semen and hyaluronidase concentration.

Influence of Cell Concentration on Respiration Rate of Sperm.

Conclusions1. At sperm concentrations from 1 to 6 billion cells per cc an increase in concentration was accompanied by a decrease in sperm respiration rate. At concentrations of 0.2 to 1 billion sperm per cc this effect of concentration was not invariably observed. 2. In contrast with the behavior of sperm cells, respiration rates of yeast cells remained constant at concentrations from 1 to 8 billion cells per cc. 3. The effect of sperm concentration on sperm respiration rate does not appear to be due to changes in pH, diminished supply of O2 to individual cells, an enzyme, or products of metabolism. 4. When comparisons are to be made of relative respiration rates of semen samples, the samples must be adjusted to the same level of sperm concentration.