Response to capacitating stimuli indicates extender-related differences in boar sperm function.

Spermatozoa, especially those of the porcine species, are highly susceptible to in vitro chilling and ageing. Extenders are continuously developed to protect boar spermatozoa from chilling injury. New semen extenders and other modified preservation strategies require sensitive testing for essential sperm functions. The key process on the pathway of fertilization is capacitation. The aim of the present study was to examine whether the specific response to capacitating stimuli is sensitive enough to indicate different preservation capacities of extenders during hypothermic storage of boar spermatozoa. Semen was diluted in Beltsville Thawing Solution (BTS) and Androstar Plus and kept for 3 h at 22°C or stored at 17°C, 10°C, and 5°C. Semen was analyzed at 24 and 96 h of storage. Motility and membrane integrity remained at high levels, except for lower values when stored in BTS at 5°C. Washed subsamples were incubated in capacitating medium (Tyrode) and control medium and were assessed for intracellular calcium concentration and integrity of plasma membranes using a flow cytometer. On the basis of the loss of low-calcium live cells in a kinetic approach, the specific response to capacitation stimuli was determined. There was a higher loss of response in semen stored hypothermically in the standard extender BTS compared to Androstar Plus. Assessment of the extent of phospholipid disorder under capacitating and control conditions by use of merocyanine staining did not reveal any significant extender-related differences. A field insemination trial with 778 sows was performed to relate in vitro results to fertility. Fertility parameters did not differ in semen stored up to 48 h at 10°C in Androstar Plus compared to controls stored at 17°C in BTS. In conclusion, assessment of specific reactivity to capacitating stimuli appears to be a sensitive tool for detection of extender-dependent alterations in functionality of chilled boar spermatozoa.

Binding of boar spermatozoa to oviductal epithelium in vitro in relation to sperm morphology and storage time.

In vitro short-term storage of boar semen for up to 72 h before insemination negatively affects fertility, but this often remains undetected during semen quality assessment. One important sperm function is the ability to form the functional sperm reservoir in the oviduct. In the present study, we used the modified oviductal explant assay to study sperm binding to oviductal epithelium in vitro in diluted boar semen stored for 24 or 72 h. First, we determined the kinetics of in vitro sperm binding to oviductal epithelium in relation to co-incubation time of sperm and oviductal tissue pieces. Then, we studied how the binding of sperm to oviductal epithelium was affected by in vitro semen storage and by differences among individual boars. Sperm binding after different incubation times was significantly higher when semen was stored 24 h than after 72-h storage (P < 0.05), and peaked at 30-90 min of incubation. Sperm binding differed between boars (n = 44), and was negatively correlated to the percentage of sperm with cytoplasmic droplets (R = -0.51, P < 0.001). There were no significant changes in motility, acrosome integrity and propidium iodide stainability during the 72-h storage period. However, sperm-binding indices were significantly lower after 72 h in vitro storage than after 24-h storage in sperm from boars with normal semen quality (P < 0.05); in contrast, the binding capacity of sperm from boars with higher percentages of morphologically altered sperm remained at a low level. The sperm-binding capacity of sperm from four of the five boars with known subfertility was lower than the mean binding index minus one standard deviation of the boar population studied here. It is concluded that changes in the plasma membrane associated with in vitro ageing reduce the ability of stored boar sperm to bind to the oviductal epithelium. This study shows the potential of sperm-oviduct binding as a tool to assess both male fertility and changes in sperm function associated with in vitro ageing.

Importance of sperm-binding assays for fertility prognosis of porcine spermatozoa.

There has been a considerable effort to establish correlations between the outcome of in vitro sperm-binding assays and the fertility achieved by individual males under conditions of commercial AI. During passage through the oviduct, a fertilizing spermatozoon has to bind to and interact with several targets. Generally, it is assumed that these interactions can be mimicked by in vitro binding assays. However, there is little evidence that assays based on zona binding, zona penetration, or IVF: (a) have been adequately validated; (b) provide data with a high degree of correlation to a boar of average fertility; (c) provide accurate predictions as to pregnancy rate and litter size from a given boar when used for commercial AI. This is due partly to the variability in measurements of pregnancy rate and litter size in a commercial setting and partly to the fact that sperm fertility is multifactorial. A recently developed in vitro test is based on the fact that spermatozoa bind in vivo to oviduct epithelium, creating a functional sperm reservoir, and that fertilization-competent spermatozoa are released in a time-dependent manner from these cells. Mating or insemination occurs usually hours before ovulation thus rendering such temporary sperm binding to the epithelial cells, a prerequisite for successful sperm-oocyte interaction. In vitro binding of porcine spermatozoa to explants derived from fresh oviduct epithelium may provide a useful test system to predict fertility, although detailed validation has not been published. The sperm-oviduct-binding assay tests for multifunctional characteristics of the plasma membrane and may be a valuable in vitro test to identify subfertile boars. We believe that boar subfertility might be indicated in vitro by reduced capacity of his spermatozoa to bind to oviductal cells and that this may provide information as to whether an adequate sperm reservoir will presumably be established in vivo from the sperm population that successfully has passed the barriers of the utero-tubal junction.

Requirement for an intact cytoskeleton for volume regulation in boar spermatozoa.

Osmotically induced cell swelling triggers a chain of events leading to a net loss of major cell ions and water, resulting in cell volume recovery, a process known as regulatory volume decrease (RVD). In many cell types, there is an evidence that the cytoskeleton may play a role in the initial sensing and transduction of the signal of volume change. In this study, we tested the hypothesis that an intact microfilament and microtubule network is required for volume response and RVD in boar sperm before and after capacitation treatment and whether addition of cytochalasin D and colchicine to the capacitation medium would affect volumetric behaviour. Capacitation is a series of cellular and molecular alterations that enable the spermatozoon to fertilize an oocyte. Cell volume measurements of washed sperm suspensions were performed electronically in Hepes-buffered saline solutions of 300 and 180 mosmol/kg. After exposure to hypoosmotic conditions, boar sperm showed initial swelling (up to 150% of initial volume within 5 min), which was subsequently partially reversed (to about 120-130% after 20 min). Treatment with cytochalasin D led to reduced initial swelling (1 micromol/l) and loss of RVD in washed sperm (1-10 micromol/l) and at the beginning of incubation under capacitating conditions (5 micromol/l). Short treatment with 500 micromol/l colchicine affected the volume regulatory ability in sperm under capacitating conditions but not in washed sperm. No significant differences in cell volume response were observed after subsequent addition of cytochalasin D and colchicine to the suspensions of sperm incubated for 3 h under capacitating conditions. However, the incubation under capacitating conditions in the presence of cytochalasin D led to improved volume regulation at the end of the incubation period (23%). The microfilament network appears to be important for volume regulation in washed boar spermatozoa while intact microtubules do not seem to be necessary for osmotically induced RVD. The changes in cytoskeleton microfilament organization during capacitation, possibly affecting the osmotically induced volume response, appear to occur at the later stages of capacitation, whereas changes in microtubules, related to volume regulatory ability, may be programmed within the first stages of capacitation.

Assessing in vivo fertilizing capacity of liquid-preserved boar semen according to the 'Hanover gilt model'.

The goal of this study was to determine the ability of the Hanover gilt model to assess in vivo fertilizing capacity of preserved sperm and to consider whether any modifications to this model were needed. This model evaluates the fertilizing capacity of semen based on the fertilization rate, the rate of normal embryos and the accessory sperm count of 3-5-day embryos. Its distinguishing characteristics are the use of one-time insemination of sperm in reduced numbers, of spontaneously ovulating gilts and of ovulation detection through ultrasound examination of ovaries. Reduced sperm numbers allow for an accurate evaluation of the fertilizing potential of different semen treatments, thereby avoiding the compensatory effect of doses calibrated to maximize fertility. The model's usefulness was assessed in a trial run designed to compare the fertilizing capacity of liquid boar semen diluted into two different extenders. The diluent, the boar and the backflow, had no significant effect on any of the parameters studied. Gilts inseminated less than 24 h before ovulation had a significantly higher (p < 0.01) fertilization rate and accessory sperm cell count (p < 0.05) than those inseminated more than 24 h before ovulation. Very good/good embryos from homogeneous litters (only very good/good embryos were present) had a significantly higher (p < 0.01) accessory sperm count than those from heterogeneous litters (at least one embryo was of a different quality and/or oocytes were present). Both very good/good and degenerated/retarded embryos from heterogeneous litters had low accessory sperm numbers. This suggests that accessory sperm count is significantly related to the quality of the litter, but not to the quality of the embryo within gilts. It can be concluded that the Hanover gilt model is sensitive enough to show fertility differences (in this study, those associated with in vivo ageing of semen), while using relatively few gilts and little time.

Role of quinine-sensitive ion channels in volume regulation in boar and bull spermatozoa.

The ability to reverse swelling caused by hypo-osmotic stress is an important cell function; in spermatozoa, it is likely to be of consequence during ejaculation and also during the thawing process that terminates cryopreservation. In this study, the time course of boar and bull sperm volume changes after exposure to hypo-osmotic conditions at 39 degrees C was recorded. Cell volume measurements of washed sperm suspensions were performed electronically in Hepes-buffered saline solutions of 300 and 180 mosmol kg(-1) containing 2.5 mmol K(+) l(-1). Treatment with quinine in the presence or absence of the potassium ionophore valinomycin was used to determine whether potassium channels were involved in the reversal of swelling. After exposure to hypo-osmotic conditions, both bull and boar spermatozoa showed initial swelling (up to 200% and 140% of initial volume, respectively, within 5 min), which was subsequently partially reversed (to about 150% and 120%, respectively, after 20 min). Incubation with quinine led to an increase in swelling in both species. However, bull sperm volume was already maximal (up to 294%) after 30 s and declined thereafter, whereas boar sperm volume increased slowly to a maximum of about 220% after 20 min. Valinomycin treatment caused quinine-induced swelling in bull spermatozoa to decrease rapidly to control (no quinine, no valinomycin) values, whereas in quinine-treated boar spermatozoa it had an opposite, enhancing effect. Interpreting these results in the light of data from studies by others on a variety of cell types, it is proposed that swelling-activated potassium channels are involved in regulatory volume decrease in both species of spermatozoa, but that boar spermatozoa may contain fewer swelling-activated chloride channels than do bull spermatozoa.

Storage of boar semen.

The problems, aspects and methods of liquid storage and freeze-thawing of boar semen are discussed and a review is given on examination of spermatozoa by the recent fluorescent staining methods.

Identification of embryo paternity using polymorphic DNA markers to assess fertilizing capacity of spermatozoa after heterospermic insemination in boars.

Differences in sperm fertilizing capacity of males often remain undetected by routine semen parameters. Heterospermic insemination with equal numbers of spermatozoa from 2 males is an accurate method for assessing differences in fertility. Use of heterospermic insemination depends on a reliable, efficient assay to identify paternity of conceptuses or offspring. In this study, polymorphic DNA markers amplified by PCR were tested to determine paternity of Day 5 to 6 embryos. The fertilizing capacity of 2 boars (A and B) with similar semen parameters was compared after homospermic (n=14 gilts) and heterospermic (n=11 gilts) insemination. Single AI's were performed under suboptimal conditions using 1 x 10(9) spermatozoa at 12 to 24 h before ovulation to prompt differences in fertilization and to stimulate sperm competition. The fertilization rate and the number of accessory spermatozoa were determined in Day 5 to 6 embryos. Using 5 different polymorphic DNA markers, paternity could be determined in 95.8% of the embryos. Boar B sired significantly (P<0.05) more offspring than Boar A after insemination with pooled semen, and this was reflected by a significantly (P<0.05) higher number of accessory spermatozoa following homospermic insemination with semen from Boar B, although fertilization rates did not differ between the 2 boars after homospermic insemination. The results suggest that the viability of spermatozoa in the female reproductive tract contributes to differences in fertility rates of males with similar in vitro sperm quality parameters. The number of accessory spermatozoa is a more sensitive measure of boar fertility than the fertilization rate. Polymorphic DNA markers are suitable for verification of parentage even at a very early stage of embryonic development.

Cyclical changes in sperm volume during in vitro incubation under capacitating conditions: a novel boar semen characteristic.

The osmotic reactivity of boar spermatozoa during incubation in vitro was studied using a hypo-osmotic swelling test in conjunction with electronic measurement of cell volume. Sperm populations showed fluctuations in both iso-osmotic cell volume and hypo-osmotic volume response that fitted mathematical models for periodicity. Significant differences of frequency and amplitude were observed during sperm incubation under capacitating conditions as compared with those under non-capacitating conditions. In addition, different boars showed specific differences in their fluctuation characteristics under capacitating conditions. During incubation under capacitating conditions, a decrease in osmotic reactivity was observed that correlated with a decrease in motility, while the absolute value of the earliest maximum of the osmotic-induced response correlated with an increase in the proportion of discharged acrosomes. The time course of the cyclical behaviour of osmotic reactivity may be a useful parameter for assessing boar sperm response to capacitating conditions.

Studies on a local effect of boar seminal plasma on ovulation time in gilts.

Previous studies showed that intrauterine infusion of seminal plasma at the onset of oestrus could advance ovulation in pigs, possibly to enhance the chances of fertilization by optimizing the chronological events of fertilization. This effect has been attributed to a local unilateral mechanism whereby infusion into a single uterine horn advances ovulation in the adjacent ovary. The present study was designed to elucidate possible mechanisms of local signal transduction. In a series of five experiments using 43 gilts, the ovarian response was investigated after infusion of seminal plasma at different sites of the female reproductive tract. The time of ovulation was detected sonographically at 4- or 2-h intervals. Single uterine horn infusion of 100 ml seminal plasma advanced ovulation on the ipsilateral ovary by 9.3 h (mean) compared with the contralateral ovary. Dissection of the ipsilateral isthmus abolished the unilateral seminal plasma effect. Unilateral infusion of 50 microliters or 1 ml seminal plasma or 50 microliters of the concentrated 1-10 kDa fraction in the lower isthmus was ineffective. Application of 5 ml seminal plasma into the tip of a ligated uterine horn lead to 3.6 h (mean) earlier ovulation on the adjacent ovary. In contrast, the infusion of 5 ml NaCl showed no effect. Application of 5 ml seminal plasma in the middle of the uterine horn between two ligatures was ineffective. It is concluded that, for the transduction of the local signal involved in the advancement of ovulation, contact of seminal plasma with the epithelium of the utero-tubal junction is essential.

[In vitro assessment of semen for the prediction of fertility]. / In-vitro-Beurteilung von Sperma zur Vorhersage der Fertilität.

The prediction of fertility is a primary goal in the field of reproductive medicine. The aim of the present paper is to describe the value of conventional and modern sperm analysis systems considering the process of fertilization. The classical assessment of motility and morphology enables the rough estimation of semen quality in order to select ejaculates for the use in artificial insemination. Recent methods for sperm diagnosis, such as fluorescent marking for the detection of sperm plasma membrane integrity, the hypoosmotic swelling test, and computer assisted semen analysis allow for the evaluation of a large number of spermatozoa and the assessment of sperm dynamics under in vitro-fertilization conditions. The oocyte penetration test investigates the ability of spermatozoa for capacitation, hyperactivation and acrosome reaction in vitro. The amount of specific seminal plasma proteins is related to fertility and thereby provides an additional semen evaluation method. For the use of a given semen test the specific in vitro condition has to be considered. In addition, the evaluated criteria relevant for the process of fertilization need to be defined. The combination of selected semen tests gives a higher accuracy for the prediction of fertilizing capacity compared with a single test.

Monoclonal antibodies against boar sperm zona pellucida-binding protein AWN-1. Characterization of a continuous antigenic determinant and immunolocalization of AWN epitopes in inseminated sows.

Boar spermadhesin AWN-1 is a sperm surface-associated 14.7-kDa lectin and a major protein of porcine seminal plasma. AWN-1 binds to beta-galactosides and to porcine zona pellucida glycoproteins, suggesting that this protein might play a role in the primary binding of spermatozoa to the egg's external glycoprotein matrix. We have produced a collection of murine monoclonal antibodies against purified AWN-1. Five monoclonal antibodies recognized sequential antigenic determinants. All these epitopes were located at the C-terminal region of AWN-1 (residues 109-123) by competitive ELISA using overlapping synthetic peptides that cover the complete 133 amino acid sequence of the lectin. In a structural model of spermadhesin AWN-1, the polypeptide stretch 109-123 is fully solvent-exposed, providing a reasonable explanation for its high immunogenicity. In addition to epitope mapping, we have employed anti-AWN monoclonal antibodies for immunolocalization of the protein in the genital tract of inseminated sows. Clusters of AWN epitopes were occasionally found attached to the epithelium of the uterotubal junction and the adjacent lower isthmus. However, neither AWN-1 nor other seminal plasma proteins were found in the isthmic fluid collected 10-26 h after insemination. These results suggest that the whole amount of seminal plasma proteins are absorbed by the epithelium of the female genital tract, supporting the claim that removal of seminal plasma components from spermatozoa might be a major event in both in vitro and in vivo sperm capacitation.

[Cooling of boar spermatozoa prior to freezing and post thaw quality and evaluation of membrane state using chlortetracycline (CTC) staining]. / Abkühlung von Eberspermatozoen vor dem Tiefgefrieren und Auftauqualität sowie Beurteilung des Membranzustandes mittels Chlortetrazyklin (CTC)-Färbung.

The influence of an extended holding time at room temperature (+18 degrees C) before freezing on boar sperm quality was investigated. 17 ejaculates were collected from 5 different boars by separation in sperm rich and sperm poor fraction. The ejaculate were split, diluted 1+1 with Merck I-Medium, and submitted to three different treatments before freezing: 1. Sperm rich fraction, cooling to +20 degrees C for 1.5 h and subsequent cooling to +15 degrees C for 2.5 h; 2. Sperm rich fraction, cooling to +18 degrees C for 4 h and subsequent holding time at +18 degrees C for 16 h; 3. Whole ejaculate (sperm rich fraction plus seminal plasma), cooling to +18 degrees C for 4 h and subsequent holding time at +18 degrees C for 16 h. Subjectively assessed post thaw motility (SMOT), computer-measured motility (CMOT), and acrosome integrity (NAR), assessed by phase contrast microscopy were significantly (p < 0.05) higher after extended holding time (procedure 2 and 3) compared to short holding time (procedure 1). The exposure to seminal plasma during holding had no significant effect. Chlortetracyclin (CTC) staining of sperm membranes gave no reliable information in the presence of an EDTA-containing preservation medium, used routinely in the preservation process.

LH profile and advancement of ovulation after transcervical infusion of seminal plasma at different stages of oestrus in gilts.

The influence of a transcervical infusion of seminal plasma on preovulatory LH profiles and the advancement of ovulation after seminal plasma infusion for different times during oestrus were investigated using the single uterine horn infusion technique (Mariensee model), in combination with transcutaneous sonographic monitoring of the ovaries. Preparative surgery in 23 German Landrace gilts comprised the detachment of the left uterine horn from the corpus, leaving the caudal end open to the peritoneal cavity but sealing the corpus wound. In six gilts fitted with a permanent jugular vein catheter the patent horns were administered a transcervical infusion of seminal plasma (n = 5 cycles) or PBS (n = 4 cycles) immediately after the detection of oestrus by a teaser boar. In addition, 17 non-catheterized gilts received infusions of seminal plasma either 0 h (n = 3 gilts), 16 h (n = 7 gilts) or 24 h (n = 7 gilts) after the detection of oestrus. Seminal plasma infusion at the onset of oestrus provoked ovulation in the ipsilateral ovary of the treated horn 8.5 +/- 0.9 h earlier than in the contralateral (control) ovary. Seminal plasma did not influence the LH profile compared with PBS (P > 0.05), but shortened the interval between the LH peak and ipsilateral ovulation to 23.4 +/- 4.0 h compared with 31.8 +/- 3.4 h in the contralateral ovulation (P < or = 0.01). Infusion 16 h after the onset of oestrus reduced the effect to 4.6 +/- 3.8 h with a wide range of 0-8 h (P < 0.01). The effect was more pronounced in gilts with long intervals between the onset of oestrus and contralateral ovulation compared with earlier ovulation on the control ovary. Seminal plasma infusion less than 16 h before contralateral ovulation and 24 h after the detection of oestrus had no effect. It is concluded that transcervical infusion of seminal plasma early in oestrus synchronizes the variable intervals between the onset of oestrus and ovulation in sows by a locally active mechanism.

Advanced ovulation in gilts by the intrauterine application of a low molecular mass pronase-sensitive fraction of boar seminal plasma.

The shortening of the time interval between the onset of oestrus and ovulation in sows by the transcervical administration of seminal plasma was investigated in 23 German Landrace gilts, using the technique of single horn infusions (Mariensee model) in combination with the transcutaneous sonographic monitoring of ovaries. Preparative surgery comprised the detachment of the left uterine horn from the corpus, leaving the caudal end open to the peritoneal cavity but sealing the corpus wound. The left ovary was loosely tied to the ventral abdominal wall for better sonographic distinction. The animals were used in two to four consecutive cycles. After detection of oestrus by the teaser boar, the patent (right) horns were filled by transcervical infusion of 100 ml of a variety of test solutions. Ovulation was probed by transcutaneous sonography at intervals of 4 h thereafter. Native seminal plasma provoked ovulation in the ipsilateral ovary of the treated horn 10.7 h earlier than in the contralateral ovary. This effect was reduced to 7.3 h after charcoal treatment of seminal plasma; addition of 10 micrograms oestradiol restored the effect in full, while 10 micrograms of oestradiol in PBS shortened the time interval to only 3.3 h versus the control ovary. Little effect was seen with oestrone sulfate, none with prostaglandins in PBS or with PBS alone. The preliminary characterization of the nonsteroidal component of seminal plasma advancing ipsilateral ovulation after transcervical infusion suggests a proteinaceous nature. The activity resides in the 1-10 kDa fraction separated by ultrafiltration and is lost after treatment with pronase.

Effect of time of insemination relative to ovulation on fertility with liquid and frozen boar semen.

Precise data on fertility results following peri- and postovulatory insemination in spontaneously ovulating gilts is lacking. Using transcutaneous sonography every 4 h during estrus as a tool for diagnosis of ovulation, the effects of different time intervals of insemination relative to ovulation were investigated with liquid semen (Experiment 1, n=76 gilts) and frozen semen (Experiment 2, n=80 gilts). In Experiment 3 (n=24 gilts) the number of Day-28 embryos related to the various intervals between insemination and ovulation was determined after the use of liquid semen. Using liquid semen the fertilization rates based on Day-2 to Day-5 embryos and the number of accessory spermatozoa decreased significantly in gilts inseminated with 2 x 10(9) spermatozoa per dosage in intervals of more than 12 h before or more than 4 h after ovulation. In the time interval 4 to 0 h before ovulation, comparable fertilization rates were obtained using frozen semen (88.1%) and liquid semen (92.5%). Fertilization rates and numbers of accessory spermatozoa decreased significantly when gilts were inseminated with frozen semen more than 4 h before or 0 to 4 h after the detection of ovulation. The percentage of Day-28 embryos was significantly higher following preovulatory insemination compared to inseminations 0 to 4 h and 4 to 8 h after ovulation. It is concluded that the optimal time of insemination using liquid semen is 12 to 0 h before ovulation, and 4 to 0 h before ovulation using frozen semen. The results stress the importance of further research on sperm transport and ovulation stimulating mechanisms, as well as studies on the time of ovulation relative to estrus-weaning intervals and estrus duration.

The initial fertilizing capacity of longerm-stored liquid boar semen following pre- and postovulatory insemination.

In pigs, high variation is seen in the duration of estrus and in the time of ovulation. This is one of a wide range of factors not related to semen quality, which possibly influences the results of field insemination trials. Experiment 1 (n=81 gilts) was performed to determine the influence of the time of ovulation on the fertilizing capacity of liquid boar semen stored up to 118 h. The objective of Experiment 2 (n=102 gilts) was to study the fertilizing potential of semen stored up to 120 h in 2 different extenders, Androhep and Beltsville Thawing Solution (BTS), by means of postovulatory AI. Inseminations were performed 0 to 4 h after ovulation in order to standardize the trial conditions. Fertilization rates based on Day-2 to Day-4 embryos, and the number of accessory spermatozoa per zona pellucida did not differ between semen stored for 0 to 48 and 48 to 87 h in gilts ovulating within 12 after insemination (Experiment 1). Gilts with an interval of 12 to 24 h between AI and ovulation had lower fertility results using semen stored for more than 48 h. A further decrease was observed when semen storage exceeded 87 h in those gilts ovulating later than 24 h after insemination. The time of ovulation has to be considered as being a major factor of variation in the fertility results of AI trials. In Experiment 2, fertilization rates and numbers of accessory spermatozoa decreased between semen stored for 0 to 24 and 24 to 48 h in BTS, and between semen stored for 0 to 24 and 48 to 72 h in Androhep. Significant differences in fertility between diluents were seen only when using semen stored for more than 96 h, with semen extended with Androhep giving the higher results. The results indicate that the decrease in fertilizing capacity due to in vitro aging of spermatozoa cannot be prevented even during the first days of storage.

[Field studies of the effect of sperm motility and morphology on the fertility of boars used for insemination]. / Spermienmotilität und -morphologie in ihrer Auswirkung auf die Fruchtbarkeit von Besamungsebern in Feldversuchen.

Based on 2127 first services in a field trial, the influence of sperm motility and morphology on the fertility of ten AI boars was investigated using semen stored for three and five days. Depending on sperm morphology the farrowing rate differed by up to 31% and the litter size differed by up to 3.4 piglets. Sperm motility and morphology are useful parameters in selecting sires for AI, especially in the case of inseminations with semen stored long-term.

[The sperm compatibility of lincospectin in the framework of the Federal Republic of Germany customary bull sperm preservation methods with the use of a Tris egg yolk thinner (clinical short report)]. / Untersuchungen zur Spermienverträglichkeit von LINCOSPECTIN im Rahmen des in der Bundesrepublik Deutschland üblichen Rindersperma-Konservierungsverfahrens unter Verwendung eines Tris-Eidotter-Verdünners (Klinische Kurzmitteilung).

Regulations considering the inner-community-exchange of frozen bovine semen prescribe the use of lincomycin and spectinomycin (LINCOSPECTIN) and streptomycin-penicillin as antibiotics in the diluted semen. A susceptibility test with Tris-egg yolk-freezing medium, mostly used in Germany, demonstrated that lincospectin in a concentration of 450 micrograms/ml diluted semen had no detrimental spermatologic effect on sperm function.