http://directory.umm.ac.id/Data%20Elmu/jurnal/A/Animal%20Reproduction%20Science/Vol60-61.Issue1-4.Jul2000/1971.pdf

The causes of reduced fertility with cryopreserved semen

http://directory.umm.ac.id/Data%20Elmu/jurnal/A/Animal%20Reproduction%20Science/Vol62.Issue1-3.Aug2000/2025.pdf

Basic aspects of frozen storage of semen

The objective of the research described was to devise an efficient procedure to cryopreserve in vitro-matured bovine oocytes, using in vitro fertilization (IVF) and development of resultant zygotes into blastocysts as criteria of oocyte survival. Oocytes at metaphase II were found to be extremely sensitive to chilling. Cooling them to O degrees C for as little as 5 sec significantly decreased their capability to cleave and develop further after IVF; after 80 sec at 0 degrees C, only approximately 10% of chilled oocytes developed into blastocysts. Oocytes were also adversely affected by brief exposures to 4 M and 5.5 M ethylene glycol (EG) solutions supplemented with sucrose; after being suspended in either of these EG solutions in plastic straws and plunged directly into liquid nitrogen (LN2), few of the oocytes were fertilized and developed. To "outrace" chilling injury, oocytes contained in < 1 microliter of EG solution were placed onto electron microscope grids and plunged directly into N2 slush or LN2. After such ultra-rapidly cooled oocytes were warmed, 30% of them cleaved after IVF, and half of these developed into blastocysts-- survival rates equivalent to those for oocytes that had been exposed to EG without any cooling. This method offers promise as a novel way to cryopreserve bovine oocytes.

/pdf/development-into-blastocysts-of-bovine-oocytes-cryopreserved-2w1ugynzlg.pdf

Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling.

Reproducing the complex process of spermatogenesis in vitro might lead to the development of new diagnostic and therapeutic techniques for male infertility. Takehiko Ogawa and colleagues have now established in vitro organ culture conditions that can support the production of fertile sperm from spermatogonia of neonatal mice. Spermatids and sperm that were derived in vitro produced healthy and fertile mice. In addition, neonatal testis tissues that were cryopreserved for several months resumed complete spermatogenesis in vitro on thawing. The organ culture method is simple and, with further refinements, could be applicable to a variety of mammalian species. This work suggests that cryopreservation of the testis tissue of paediatric cancer patients could become a practical way of ensuring future fertility. Reproducing the complex process of spermatogenesis in vitro might lead to the development of new diagnostic and therapeutic techniques for male infertility. This study establishes in vitro organ culture conditions that can support complete spermatogenesis in mice. The in-vitro-derived spermatids and sperm produced healthy and fertile mice, and testis tissue fragments used as a starting material for in vitro spermatogenesis could be cryopreserved for months and then resumed full spermatogenesis in vitro. Spermatogenesis is one of the most complex and longest processes of sequential cell proliferation and differentiation in the body, taking more than a month from spermatogonial stem cells, through meiosis, to sperm formation1,2. The whole process, therefore, has never been reproduced in vitro in mammals3,4,5, nor in any other species with a very few exceptions in some particular types of fish6,7. Here we show that neonatal mouse testes which contain only gonocytes or primitive spermatogonia as germ cells can produce spermatids and sperm in vitro with serum-free culture media. Spermatogenesis was maintained over 2 months in tissue fragments positioned at the gas–liquid interphase. The obtained spermatids and sperm resulted in healthy and reproductively competent offspring through microinsemination. In addition, neonatal testis tissues were cryopreserved and, after thawing, showed complete spermatogenesis in vitro. Our organ culture method could be applicable through further refinements to a variety of mammalian species, which will serve as a platform for future clinical application as well as mechanistic understanding of spermatogenesis.

http://basicmed.med.ncku.edu.tw/admin/up_img/1220-1.pdf

In vitro production of functional sperm in cultured neonatal mouse testes

The genetic improvement of agriculturally important species and disease control are of fundamental importance to the success of a sustainable agri-food industry. In this sense, artificial insemination (AI) is arguably the most important tool contributing to the advancement of modern animal production. Through AI, 1 ejaculate from a genetically superior male can be used to impregnate multiple females to maximize the distribution of agriculturally favorable genes. As well, AI eliminates physical contact between animals, thus limiting the spread of sexually transmitted diseases. Successful semen cryopreservation enhances these advantages of AI over natural breeding. Long-term storage facilitates semen transport over distances, permits the quarantine of semen, and enables extended use of superior germplasm, even after the sire’s death. For agriculturally important animals, semen cryopreservation is an established industry worldwide, particularly for dairy cattle. Genome resource banking to preserve the biodiversity of endangered species or valuable transgenic lines also would benefit from sperm cryopreservation. For many mammals; however, effective semen cryopreservation is not a reality because a large number of sperm are apparently infertile following freezing and thawing. Compared to fresh, 8 times more cryopreserved bovine sperm were required to achieve equivalent fertilization rates in vivo (Shannon and Vishwanath, 1995). Although AI in pigs with fresh, cooled semen is increasingly popular, the fertility of cryopreserved semen remains about half that of fresh semen (Crabo, 1991). Following either transuterine or oviductal insemination of superovulated ewes, conception rates and percentage of fer-

https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.1939-4640.2000.tb03268.x

Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon.

Lipid composition and thermotropic phase behavior of boar, bull, stallion, and rooster sperm membranes.

Effects of cryopreservation procedures on sperm membranes

The purpose of this study was to evaluate effects of cooling and rewarming on the meiotic spindle apparatus of bovine oocytes In experiment 1, in vitro-matured bovine oocytes were either maintained at 39 degrees C or cooled abruptly to 4 degrees C or approximately 25 degrees C Immunohistochemical and DNA staining for visualization of microtubules and chromosomes, respectively, revealed an anastral, barrel-shaped spindle in bovine oocytes Exposure to 4 degrees C for 10-20 min caused complete disappearance of the spindle Some chromosome dispersion occurred after 60 min at 4 degrees C After exposure to approximately 25 degrees C for 30 min, 90% of oocytes appeared abnormal, having either an abnormal spindle or no spindle In experiment 2, oocytes cooled to either approximately 25 degrees C or 4 degrees C for 30 min were rewarmed directly or in steps for 15 or 60 min Spindles did not return to normal in most oocytes regardless of cooling temperature or rewarming scheme Step-wise rewarming was no more beneficial than direct rewarming More of the oocytes rewarmed directly contained dispersed chromosomes as time at 39 degrees C increased

Effects of cooling and rewarming on the meiotic spindle and chromosomes of in vitro-matured bovine oocytes

Factors affecting low temperature survival of mammalian oocytes

Bovine oviductal fluid (OF) was collected and analyzed throughout the estrous cycle, and the capacity of the protein and lipoprotein components to support cholesterol efflux from bovine sperm was evaluated. Blood was collected and assayed for progesterone (P4) to monitor the estrous cycle. Protein and lipoprotein separation was achieved by density gradient centrifugation. Two major bands were identified. The first (1.056 less than delta 20 less than 1.140 g/ml) corresponded to bovine and rabbit plasma high-density lipoprotein (HDL) based on distribution in the density gradient and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The second band (1.235 less than delta 20 less than 1.243 g/ml) consisted predominantly of oviductal fluid albumin (OFA). Oviductal fluid protein concentration increased as serum P4 decreased around the time of estrus. Mean OF protein concentration was 21.3 mg/ml when serum P4 was lower than 0.5 ng/ml and 6.9 mg/ml when serum P4 was greater than 0.5 ng/ml. An inverse log relationship was found between HDL protein concentration and serum P4. Unesterified cholesterol (UC), cholesteryl ester, and phospholipid (PL) content of HDL for HDL protein concentrations of 3-56.1 micrograms/ml were 1.35-46.2 micrograms/ml, 1.91-44.48 micrograms/ml, and 1.69-59.8 micrograms/ml, respectively. Phosphatidylcholine and -ethanolamine were the major PLs present in the HDL fraction and their molar ratio (4:1 mol/mol) was relatively constant through the estrous cycle. The OFA fraction of the same samples accounted for more than 90% of total protein and for most of the variation in OF protein. To determine the ability of OF components to serve as sperm cholesterol acceptors, OF samples were incubated 1:1 (v/v) with and without 4 X 10(8) bovine sperm in 1.0 ml of modified Tyrode's solution and OF for 2 hr at 39 degrees C. After incubation, HDL and OFA fractions were isolated and analyzed for changes in protein and lipid content. After OF, samples were incubated with sperm, an increase in UC was found in the HDL fractions. UC in HDL increased by 12.1 +/- 1.0 micrograms/ml (means +/- SE) when serum P4 was less than or equal to 0.5 ng/ml. For samples corresponding to higher serum P4, the increase in UC was 3.60 +/- 0.89 micrograms/ml. Values for UC in HDL were corrected for the contribution of UC from OFA of OF samples. Cholesterol efflux from sperm has been implicated in the process of sperm capacitation. These results indicate that HDL from OF is elevated during the follicular phase of the estrous cycle and can serve as an acceptor for bovine sperm cholesterol.

John E. Parks

Papers

Lipid composition and thermotropic phase behavior of boar, bull, stallion, and rooster sperm membranes.

Effects of cryopreservation procedures on sperm membranes

Effects of cooling and rewarming on the meiotic spindle and chromosomes of in vitro-matured bovine oocytes

Factors affecting low temperature survival of mammalian oocytes

Bovine oviductal fluid components and their potential role in sperm cholesterol efflux.