Semen from rainbow trout produced using cryopreserved spermatozoa is more suitable for cryopreservation

Oocytes from three female rainbow trout Oncorhynchus mykiss were inseminated separately with untreated or cryopreserved semen, which had been produced using either untreated (three males) or cryopreserved (three males) spermatozoa. In half of variants, the cryopreservation did not significantly affect fertilization efficiency. Regardless of whether the sperm donors were produced from cryopreserved or intact semen, insemination of oocytes with their intact sperm resulted in the same percentage of eyed embryos (94.4 and 94.3%, respectively). When eggs were inseminated with cryopreserved semen, the use of sperm from males produced with cryopreserved spermatozoa resulted in a significantly higher percentage of eyed eggs than in case of donors produced with intact sperm (89.6 and 81.7%, respectively). The production of rainbow trout using cryopreserved sperm does not appear to negatively affect reproductive abilities of male progeny and semen from donors, which were produced using cryopreserved sperm, is more suitable for cryopreservation than the semen from donors produced with intactspermatozoa.     

Apyrene sperm from the triploid donors restore fecundity of cryopreserved semen in Bombyx mori

Female moths of Bombyx mori were artificially inseminated with cryopreserved semen. The fertility of inseminated females varied from 0% to 76.9% depending on the strain. Addition of fresh semen from triploid males, which are infertile but whose semen includes intact apyrene sperm, greatly improved fecundity of cryopreserved semen from normal males. Frozen apyrene sperm from the triploid donors also improved the fecundity of females, inseminated with cryopreserved normal semen, but less than fresh semen from triploid males. Fertilization success in B. mori requires the presence of both, intact eupyrene and apyrene sperm. Our results show that eupyrene sperm tolerate the cryopreservation process better than apyrene sperm. Hence, we recommend to add apyrene sperm from the triploid donors as helper sperm routinely to cryopreserved semen in artificial insemination. This may advance the application of cryopreservation as a routine technique to maintain silkworm resources. The technique may also be applicable to other moth and butterfly species which, like B. mori, possess eupyrene and apyrene sperm.     

Application of artificial insemination technique to eupyrene and/or apyrene sperm in Bombyx mori

The silkworm, Bombyx mori, has a dimorphic sperm system. The eupyrene sperm is the sperm to fertilize eggs and the apyrene sperm plays a crucial role for assisting fertilization. Heat-treated (33 degrees C for 96h) Daizo (DH) males, one of the strains in the silkworm, produce only eupyrene sperm, while in triploid males only apyrene sperm are functional. Though both types of males are found to be sterile, double copulation of the two males with a single female greatly increases fertility. Here we examined the fertilizing ability of eupyrene and apyrene sperm by means of an artificial insemination technique previously established in B. mori. Neither the eupyrene sperm collected from DH males, nor the apyrene sperm from triploid males have the ability to fertilize eggs. Artificial insemination with the mixture of eupyrene and apyrene sperm leveled up the frequency of fertilized eggs to more than 80%. When cryopreserved DH sperm (eupyrene sperm) were subjected to the same experiment, more than 95% fertilized eggs were obtained. These results confirmed that apyrene sperm play an important and indispensable role in fertilization in B. mori. Separate collection of functional eupyrene sperm and functional apyrene sperm and success of fertilization by means of the artificial insemination technique are applicable for further studies to elucidate the function of apyrene sperm.     

Freezing tolerance in embryos and spermatozoa of the sea urchin

Late embryos of the sea urchin survive freezing, at least for a short period of time, at ?196 °C in the presence of a cryoprotectant. The freezing tolerance in glycolated embryos is greater in advanced developmental stages. High rates of both cooling and warming during a freezethaw sequence were more dangerous in one-cell embryos than in late ones. Both ethylene glycol and DMSO exerted a significant protection against freezing injury on embryos in all the stages after fertilization, but not on unfertilized egg cells. Cryopreservation of sea urchin sperm at ?196 °C in the presence of 1.5 m ethylene glycol has been achieved for 3 days. The fertilizability of eggs inseminated by frozen-thawed sperm was more than two-thirds. Nearly all the eggs thus fertilized developed to normal gastrulae.     

Artificial insemination using trypsin-treated sperm in the silkworm,Bombyx mori

A new method was developed for artificial insemination of the silkworm. Virgin females artificially inseminated by this method oviposited fertilized eggs at a rate almost similar to that of females mated with males. Using inactive sperm collected from the seminal vesicles, we confirmed the previous finding by Omura, S., 1936a, Artificial insemination of Bombyx mori, Journal of Faculty of Agriculture, Hokkaido Imperial University 38, 135-150, and Omura, S., 1938, Studies of the reproductive system of the male of Bombyx mori II, Post-testicular organs and post-testicular behaviour of the spermatozoa, Journal of Faculty of Agriculture, Hokkaido Imperial University 40, 129-170, that the sperm of B. mori requires the secretion of the glandula prostatica for activation. Sperm also could be activated by trypsin. At an optimal concentration, 0.3 &mgr;g trypsin/ml in 50% semen solution, the fertilization rate as well as the number of eggs oviposited was almost equivalent to that obtained in normally mated moths. These results may contribute not only to basic studies on fertilization and reproduction in Lepidoptera but also to the development of long-term preservation of genetic resources by using cryopreserved sperm of B. mori and other Lepidoptera insects.     

Fertilized eggs obtained from transplantation of frozen ovaries and parthenogenesis in combination with artificial insemination of frozen semen of the silkworm,Bombyx mori

A reliable method is reported for the long-term preservation of ovaries and spermatozoa of the silkworm (Bombyx mori). Three studies are presented. In the first, ovaries were removed from larvae at either 3rd, 4th, or 5th instar, cryopreserved, and stored in liquid nitrogen. Thawed ovaries were transplanted to surgically castrated female larvae at the same or a different developmental stage. The highest percentage of recipient females producing eggs resulted into either 3rd or 4th instar larvae (respectively, 22.1 and 8.7%). Similarly, the highest levels of other measurements of successful cryopreservation and transplanted ovary, and number of eggs laid, occurred with the same combination of donor and recipient developmental stages. Other combinations of ovary/recipient developmental stages yielded lower results. In the second experiment, semen was collected from male moths, cryopreserved, and then thawed semen was diluted with trypsin solution and artificially inseminated into females obtained from the best conditions of first experiment. A small percentage of inseminated moths laid eggs (8-10.3%) compared to that of controls (100%). In addition, the fertility of eggs from experimental moths was lower than that of control females (respectively, 40.3-88% and 97.5%). In the third experiment, eggs were surgically removed from ovarian tubules of moth following transplantation of thawed ovaries and subjected to parthenogenetic activation and artificial hatching. As expected, all resulting moths were female and, following natural mating or artificial insemination with thawed semen, yielded normal offspring at high rates.     

昆明小鼠精子冷冻的研究（简报）

胚胎工程技术是动物品种、品系培育,种质资源保存及转基因动物制备、保种的重要手段。配子的冷冻保存技术目前广泛应用于胚胎工程。和胚胎冷冻相比小鼠精子冷冻技术方便、高效尤其适用于转基因及突变系小鼠的保种。成功的精子冷冻要求复苏后通过体外受精(IVF)获得胚胎,再移植入受     

Semen cryopreservation of small abalone (Haliotis diversicolor supertexa)

Methods for cryopreserving spermatozoa and maximizing fertilization rate in Taiwan small abalone, Haliotis diversicolor supertexa, were developed. The gametes (spermatozoa and eggs) of small abalone were viable 3 h post-spawning, with fertilization, and development rate decreasing with time. A minimum of 10(2) cell/ml sperm concentration and a contact time of 2 min between gametes is recommended for artificial insemination of small abalone eggs. Eight cryoprotectants, dimethyl sulfoxide (DMSO), dimethyl acetamide (DMA), ethylene glycol (EG), propylene glycol (PG), butylene glycol (BG), polyethylene glycol, glycerol and methanol, were tested at concentrations between 5 and 25% to evaluate their effect on motility of spermatozoa exposed to cryoprotectant for up to 60 min at 25 degrees C before freezing. The least toxic cryoprotectant, 10% DMSO, was added to artificial seawater (ASW) to formulate the extender for freezing. Semen was diluted 1:1 with the extender, inserted into 1.5 ml microtubes and frozen using a cooling rate between -3.5 and -20 degrees C/min to various transition temperatures (0, -30, -60, -90 and -120 degrees C), followed by transfer and storage in liquid nitrogen (-196 degrees C). The microtubes were thawed from +45 to +145 degrees C/min. Spermatozoa, cooled to -90 degrees C at a cooling rate of -12 or -15 degrees C/min and then immersed in liquid nitrogen, had the best post-thaw motility. Post-thaw sperm motility was markedly reduced compared to fresh sperm. More frozen-thawed spermatozoa are required to achieve fertilization rates comparable to those achieved using fresh spermatozoa.     

Fertility of cooled and frozen rabbit sperm measured by competitive fertilization

The fertility of rabbit sperm that had been cooled to 5 degrees C or frozen and thawed was determined by competitive fertilization. Treatments were identified by labeling sperm with fluorescein isothiocyanate (FITC) or tetramethylrhodamine B isothiocyanate (TRITC). Sperm from different treatments were mixed and used in a competitive insemination experiment. Does were inseminated 5, 10 or 15 h prior to ovulation. Time of ovulation was controlled by injections of luteinizing hormone. The functional sperm transport, as determined by the number of sperm transported to the site of fertilization and capable of fertilizing oocytes, was estimated by counting the total number of differently stained sperm that surrounded or fertilized each oocyte. The fertility of sperm cooled to 5 degrees C was not affected (p less than 0.05) as compared to fertility of uncooled sperm. Functional sperm transport at all times of insemination and fertilization ratio at insemination 10 or 15 h before ovulation were reduced (p less than 0.05) for frozen-thawed vs. cooled sperm. No difference in fertilization ratio (p greater than 0.05) occurred, however, when does were inseminated 5 h before ovulation. While sperm survival and capacitation time appeared to play roles in fertility of frozen-thawed sperm, the most important factor was reduced functional sperm transport. However, fertility of frozen-thawed sperm was improved when the time from insemination to ovulation was reduced.     

New aspects of boar semen freezing strategies

Although cryopreserved boar semen has been available since 1975, a major breakthrough in commercial application has not yet occurred. There is ongoing research to improve sperm survival after thawing, to limit the damage occurring to spermatozoa during freezing, and to further minimize the number of spermatozoa needed to establish a pregnancy. Boar spermatozoa are exposed to lipid peroxidation during freezing and thawing, which causes damage to the sperm membranes and impairs energy metabolism. The addition of antioxidants or chelating agents (e.g. catalase, vitamin E, glutathione, butylated hydroxytoluene or superoxide dismutase) to the still standard egg-yolk based cooling and freezing media for boar semen, effectively prevented this damage. In general, final glycerol concentrations of 2-3% in the freezing media, cooling rates of -30 to -50 degrees C/min, and thawing rates of 1200-1800 degrees C/min resulted in the best sperm survival. However, cooling and thawing rates individually optimized for sub-standard freezing boars have substantially improved their sperm quality after cryopreservation. With deep intrauterine insemination, the sperm dose has been decreased from 6 to 1x10(9) spermatozoa without compromising farrowing rate or litter size. Minimizing insemination-to-ovulation intervals, based either on estimated or determined ovulation, have also improved the fertility after AI with cryopreserved boar semen. With this combination of different approaches, acceptable fertility with cryopreserved boar semen can be achieved, facilitating the use of cryopreserved boar semen in routine AI programs.     

Cryopreservation of yellowfin seabream (Acanthopagrus latus) spermatozoa (Teleost, Perciformes, Sparidae)

The effects of both osmolality and cation in the initiation of sperm motility were examined in yellowfin seabream, Acanthopagrus latus. Various factors involved in the cryopreservation of yellowfin seabream spermatozoa on motility are discussed. Extender containing only glucose proved to be a suitable medium for freezing yellowfin seabream spermatozoa to -196 degrees C. Glycerol seems to have a direct osmotic effect on yellowfin seabream sperm cells, and it induced sperm motility before freezing and during thawing. However, this exhausted the energy needed for sperm motility for fertilization. Dimethyl sulfoxide (DMSO) proved superior to ethylene glycerol, propylene glycerol, glycerol and methanol as a cryoprotectant. Prolonged equilibration time had a detrimental effect on both prefreezing and post-thawing sperm motility. The estimated optimum freezing rate was in the range of -20 to -154 degrees C/min. More frozen-thawed than fresh spermatozoa are required to achieve comparable fertilization rates.     

Development of a cryopreservation protocol for long-term storage of black tiger shrimp (Penaeus monodon) spermatophores

The objectives of this study were to determine the effect of cryoprotectants on sperm viability and develop a freezing protocol for long-term storage of P. monodon spermatophores. Spermatophores suspended for 30 min in calcium-free saline (Ca-F saline) containing the cryoprotectants dimethyl sulfoxide (DMSO), ethylene glycol (EG), 1,2-propylene glycol (PG), formamide, and methanol at concentrations of 5, 10, 15, or 20% were studied using a modified eosin-nigrosin staining technique. The smallest reductions in apparent sperm viability occurred with DMSO; therefore, a freezing protocol was developed using Ca-F saline containing 5% DMSO. Spermatophores were cryopreserved using three protocols; cooling to a final temperature of -30, -80 or -80 degrees C and immediately stored in liquid nitrogen (cooling rates of -2, -4, -6, -8, -10, -12, -14 or -16 degrees C/min). Frozen spermatophores were thawed (2 min) at 30, 60, 70, or 90 degrees C. Successful cryopreservation of spermatophores in liquid nitrogen was achieved by a one-step cooling rate of -2 degrees C/min between 25 and -80 degrees C before storing in liquid nitrogen. Optimal thawing was in a 30 degrees C water bath for 2 min; this yielded live sperm after storage in liquid nitrogen for 210 days. Average sperm viability for fresh (97.8+/-2.9%) and cryopreserved spermatophores held for less than 60 days (87.3+/-4.1%) did not differ (P>0.05); however, that for spermatophores stored in liquid nitrogen between 90 and 210 days were lower (P<0.05) and varied from 27.3+/-3.4 to 53.3+/-4.3%. Thawed spermatophores previously held in liquid nitrogen for less than 62 days fertilized eggs (fertilization and hatching rates of 71.6-72.2% and 63.6-64.1%, respectively) at rates comparable to fresh spermatophores (70.8-78.2% and 66.3-67.8%, respectively). In conclusion, sperm within cryopreserved spermatophores stored in liquid nitrogen retained their viability for up to 210 days.     

Comparison of heterospermic and homospermic inseminations as measures of male fertility

This study attempted to determine a basis for the previously observed greater sensitivity of heterospermic tests when compared to homospermic tests for detecting differences in fertility between males. In theory, the results of heterospermic tests are an indication of the proportion of eggs fertilized per unit time whereas results of homospermic inseminations measure only the cumulative or final proportion of eggs fertilized. The fertilizing ability of sperm from males of CF1 and C57BL/6N strains of mice was compared homospermically using both relatively high and low concentrations of sperm and by measuring the proportion of eggs penetrated per unit of time. The fertilizing ability of sperm from these strains was also compared using heterospermic inseminations. When females were inseminated with a high concentration of sperm, males of both strains fertilized a high and indistinguishable percentage of eggs when examined after 30 hr. When females were inseminated with either a low concentration of sperm or when the proportion of eggs penetrated was measured at 5 hr, differences between strains of mice were distinguishable. Heterospermic insemination further magnified the observed difference between strains. The results of this study confirm that measuring the percentage of eggs fertilized per unit of time can enhance the magnitude of differences between males in fertility as compared to measuring only the final percentage of eggs fertilized. Measuring the percentage of eggs fertilized per unit of time does not, however, entirely account for the large differences observed between fertility of males when they are compared using heterospermic inseminations.     

Sperm precedence in the deer tick Ixodes dammini

ABSTRACT We determined whether female deer ticks Ixodes dammini Spielman, Clifford, Piesman & Corwin (Acari: Ixodidae) can be inseminated repeatedly and whether sperm from first or second matings take precedence in fertilizing eggs. Such information is essential to the design of attempts to reduce the fertility of these vectors of Lyme disease. Although spermatophores are present in about half of questing female ticks, they are present in virtually all those found on deer; the abundance of males on deer exceeds that of females and copulation is common. Females must be inseminated before commencing the rapid engorgement phase of feeding. Males need not be in attendance during feeding, provided that the female has been inseminated preprandially. Thus, preprandial insemination suffices to stimulate rapid engorgement, but less blood is taken than when the female is perprandially inseminated. Both types of insemination effectively fertilize eggs. Eggs from females sequentially inseminated by irradiated and non irradiated males, were fertilized mainly by sperm from the last male. Cobalt-irradiated males mate effectively and their sperm compete with those of non-irradiated males. Sperm from the second two sequential inseminations fertilize most of the eggs. By infesting deer with such irradiated male I.dammini , the abundance of these vector ticks may effectively be reduced.     

Effects of cooling and warming conditions on post-thawed motility and fertility of cryopreserved buffalo spermatozoa.

The principal objective of this study was to derive an improved procedure for cryopreservation of swamp buffalo (Bubalus bubalis) spermatozoa. Experiments were conducted to determine effects of cooling rate, intermediate plunge temperature and warming rate on motility and acrosome integrity of spermatozoa. Spermatozoa were obtained from three bulls (three ejaculates/bull) and were subjected to nine cooling conditions before being frozen in liquid nitrogen: cooling at 10, 20, or 30 degrees C/min each to -40, -80, or -120 degrees C before being plunged into liquid nitrogen. The spermatozoa frozen under a given condition were then thawed either at 1000 or 200 degrees C/min. Cooling rate, intermediate temperature and warming rate significantly affected survival of spermatozoa obtained from the three bulls. Cooling spermatozoa from 4 to -120 degrees C either at 20 or 30 degrees C/min yielded better progressive motility compared to other cooling conditions (50 versus 30%). Rapid warming was superior to slow warming. In an additional study, motility and fertility of spermatozoa frozen after being cooled to -120 degrees C at 20 degrees C and 30 degrees C/min and those frozen by a standard protocol used routinely for semen processing were assessed. Progressive motility of cryopreserved spermatozoa cooled at 20 degrees C and 30 degrees C/min was 40%, while that of spermatozoa cryopreserved using a standard protocol was 25%. A total of 178 buffalo cows were inseminated with cryopreserved spermatozoa obtained from one bull, and their pregnancy status was assessed 60 days later by rectal palpation. Out of the 60, 26 (43%) and 23 of 58 (40%) cows inseminated with sperm cooled at 20 and 30 degrees C/min, respectively, became pregnant, whereas 17 of 60 (28%) cows inseminated with sperm frozen by a standard protocol became pregnant. This study demonstrates that an effective cryopreservation procedure for buffalo spermatozoa can be derived by systematic examination of various cryobiological factors.     

Applications of cryopreserved unfertilized mouse oocytes for in vitro fertilization

Since the first successful reports into oocyte freezing, many papers concerning the cryopreservation of mouse oocytes have been published. However, a simple and practical cryopreservation method for unfertilized C57BL/6 mouse oocytes, and an IVF system using these cryopreserved oocytes have yet to be established, in spite of the fact that C57BL/6 is the prevalent inbred strain and is used for large-scale knockout programs. In this study, unfertilized C57BL/6 mouse oocytes were cryopreserved via a simple vitrification method. After warming, IVF was performed using cryopreserved unfertilized oocytes and fresh sperm, cryopreserved unfertilized oocytes and cold-stored sperm, cryopreserved unfertilized oocytes and frozen sperm (C57BL/6 strain sperm), and cryopreserved unfertilized oocytes and frozen sperm derived from GEM strains (C57BL/6 background GEM strains). Nearly all of the cryopreserved oocytes were recovered, of which over 90% were morphologically normal. Those oocytes were then used for in vitro fertilization, resulting in 72–97% of oocytes developing into 2-cell embryos. A portion of the 2-cell embryos were transferred to recipients, resulting in live young being produced from 32–49% of the embryos. In summary, we established the simple and practical method of mouse oocyte vitrification with high survivability and developmental ability and the IVF using the vitrified-warmed oocytes with fresh, cold-stored or cryopreserved sperm with high fertility.     

Birth of mice after in vitro fertilization using C57BL/6 sperm transported within epididymides at refrigerated temperatures

The transportation of cryopreserved spermatozoa is an economical, efficient, and safe method for the distribution of mouse strains from one facility to another. However, spermatozoa from some strains, including C57BL/6 (B6), are very sensitive to freezing and thawing and frequently fail to fertilize eggs by conventional in vitro fertilization methods at the recipient mouse facility. Since many genetically engineered mice have the B6 genetic background, this sensitivity poses a major obstacle to studies of mouse genetics. We investigated the feasibility of transporting spermatozoa within epididymides under non-freezing conditions. First, we examined the interval that B6 and B6D2F1 (BDF1) spermatozoa retained their ability to fertilize when stored within epididymides at low temperatures (5 degrees C or 7 degrees C). Fertilization rates were >50%, irrespective of the spermatozoa used, when epididymides were stored for 3d at 7 degrees C. B6 spermatozoa, but not BDF1 sperm, had better retention of fertilizing ability at 7 degrees C versus 5 degrees C. We then transported freshly collected B6 and BDF1 epididymides from a sender colony to a recipient colony using a common package delivery service, during which the temperature was maintained at 5 degrees C or 7 degrees C for 2d. Sufficiently high fertilization rates (68.0-77.5%) were obtained for all experimental groups, except for B6 spermatozoa transported at 5 degrees C. These spermatozoa were successfully cryopreserved at the recipient facility and, yielded post-thaw fertilization rates of 27.6-66.4%. When embryos derived from the B6 spermatozoa that were transported at 7 degrees C were transferred into recipient females, 52.7% (38/72) developed to term. In conclusion, transportation of epididymides at refrigerated temperatures is a practical method for the exchange of mouse genetic resources between facilities, especially when these facilities do not specialize in sperm cryopreservation. For the B6 mouse strain, the transportation of epididymides at 7 degrees C rather than 5 degrees C, is recommended.     

Sperm concentration and the fertilization of human eggs in vitro

The effect of sperm concentration on the fertilization of preovulatory and immature human eggs was studied in the context of an ongoing in vitro fertilization-embryo transfer (IVF-ET) program. Fertilization success was independent of the follicular recruitment protocol used, and with preovulatory eggs, was inversely related to sperm concentration over the range of 2.5 - 50 X 10(4) motile sperm/ml. Maximum fertilization (80.8%) occurred at a concentration of 2.5 X 10(4) motile sperm/ml. The incidence of polyspermic fertilization was directly related to the sperm concentration, decreasing from 5.5% at 10 X 10(4) to 0% at 1-2.5 X 10(4) motile sperm/ml. Immature eggs cultured in vitro, then inseminated, also demonstrated an inverse relationship between fertilization and sperm concentration with a maximum fertilization rate of 66.6% at 5 X 10(4) motile sperm/ml. The percentage of motile sperm in the inseminating population had no influence on fertilization rates unless the value dropped below 40%. Fertilization success using sperm from oligospermic and polyzoospermic males was also examined. In contrast to males with normal semen parameters, oligospermic males demonstrated highest fertilization success at 50 X 10(4) motile sperm/ml. The IVF of preovulatory eggs using sperm from polyzoospermic males was comparable to that for males with normal semen parameters at equivalent sperm concentrations. The implications of these findings to the application of IVF-ET technology to the infertile couple is discussed.     

Penetration of mouse eggs at various intervals after insemination as influenced by concentration of sperm and strain of male

This study was conducted to determine the extent to which the percentage of mouse eggs that were penetrated by sperm at the end of the period of sperm penetration was due to the proportion of eggs penetrated per unit of time and to the span of time of sperm penetration. Female mice of ICR strain were inseminated 1.5 hr after ovulation with 5 X 10(6) sperm/50 microliter from males of DBA/2N, CF1 or C57BL/6N strains to determine the effect of the male. To determine the effect of concentration of sperm ICR females were inseminated with 2, 4, 6, or 8 X 10(6) sperm/50 microliter from CF1 males. Females were killed at various intervals after insemination and the eggs were recovered and examined for evidence of penetration by a sperm. The time intervals from both insemination to the onset of egg penetration and from insemination to cessation of penetration were similar for the three strains of males. Throughout the period of penetration of eggs a constant percentage of eggs was penetrated per hour for a particular strain of male. The relative percentage penetrated per hour very closely approximated the relative percentage of eggs finally penetrated for each strain of male. The percentage of eggs penetrated per hour was linearly positively related to the concentration of sperm inseminated. The final percentage of eggs penetrated depended primarily on the rate at which the eggs were penetrated during the period of sperm penetration and not on the length of the period of egg penetration which was constant.     

Rooster spermatozoan motility,forward progression,and fertility after storage at 25, 5, or −196 °C in various extenders

Rooster spermatozoa were stored at 25, 5, or ?196 °C in either TC199, a pyruvate-lactate mouse ova culture medium, or as undiluted semen. There was a linear decrease in percent of motile sperm during storage at 25 or 5 °C in all cases, and a curvilinear decrease with increasing storage times at ?196 °C. Percent of motile sperm present after increasing storage time suggested pyruvate-lactate is a better extender than TC199 at the three storage temperatures studied. Pullets inseminated with 1 × 10⁸ motile sperm using fresh sperm diluted in TC199 or pyruvate-lactate, or stored 24 hr at 5 or ?196 °C produced 68.7, 74.1, 20.6, and 10.8% fertile eggs, respectively. The differences in fertility between controls or between samples stored at 5 and ?196 °C were not significant. However, fertility from sperm stored at 5 and ?196 °C was significantly lower (p < .05) than both control groups. Thus, it can be concluded that TC199 or pyruvate-lactate may be used to dilute fresh rooster semen collections prior to insemination. In contrast, fertility of rooster sperm is not satisfactorily maintained after 5 or ?196 °C storage for 24 hr in a pyruvate-lactate extender.