This investigation comprises three trials. Trial 1 consists of an in vitro comparison of three semen extenders: two egg yolk based (customized Tris-egg yolk-glycerol and Triladyl ® ), the third (AndroMed ® ) soybean lecithin based. With regard to post-thaw motility, the phytoextender AndroMed ® proved to be superior (59 ± 3% v. 53 ± 2% and 53 ± 2%, P < 0.05). It had earlier been shown that addition of the commercial prostaglandin F 2α preparation Dinolytic ® before freezing compromises post-thaw motility; therefore, in Trial 2, Dinolytic ® was added after thawing. Frozen-thawed spermatozoa tolerated addition of Dinolytic ® at a concentration of 30% (v/v). In Trial 3, cows were inseminated using straws in which diluted semen and Dinolytic ® were frozen in the same straw, separated by an air bubble, so intermingling could only take place in the course of insemination. Pregnancy rates at Dinolytic ® dosages of 0%, 30% or 60% amounted to 44%, 41% and 56%, respectively ( P > 0.05), a result that encourages a large-scale field study, which is envisioned.Keywords: cattle, artificial insemination, spermatozoan motility, semen extender, prostaglandin F 2α ImplicationsPregnancy rates of well below 50% are common in most dairy operations. There is published evidence that addition of prostaglandin F 2α (PGF 2α ) to semen may improve conception rates, presumably by enhancing uterine contractility, and thus sperm transport to the site of fertilization. This could be particularly beneficial when inseminating with small sperm numbers, for example, sexed semen. The present study establishes preconditions (suitable semen extender, PGF 2α concentration tolerated by sperm cells and invention of a two-column straw artificial insemination technique) for implementation of a large-scale field investigation. IntroductionThe present study addresses the establishment of preconditions for a large-scale field trial aimed at verifying results reported by Gabriel et al. (2011), suggesting that intrauterine administration of prostaglandin F 2α (PGF 2α ) at the time of insemination improves the pregnancy rate in dairy cows. In that trial, i.m. injection of either Dinoprost (Dinolytic ® , Dinoprost-Trometamol; Zoetis, Berlin, Germany) or the synthetic PGF 2α analog cloprostenol was ineffective, and so was intrauterine infusion of cloprostenol. Intrauterine infusion of Dinoprost, however, brought about an increase in the proportion of cows falling pregnant. At slightly >220 cows/ treatment, the effect was not statistically significant (P = 0.12). Before initiating a new study involving substantially more animals per treatment, the practicability of the treatment was improved by incorporating Dinoprost into the insemination straw instead of having to administer it separately as practiced by Gabriel et al. (2011). The first intention of the present study was to compare various semen extenders on the basis of post-thaw motility. As a second step the concentration of Dinolytic ® that is tolerated by sperm cells was determined. Previous in vitro exp...

A field study was conducted aimed at (i) evaluating the practicability of a fixed-time insemination regime for medium-sized dairy operations of north-western Germany, representative for many regions of Central Europe and (ii) substituting hCG for GnRH as ovulation-inducing agent at the end of a presynch or ovsynch protocol in an attempt to reduce the incidence of premature luteal regression. Cows of two herds synchronized by presynch and two herds synchronized by ovsynch protocol were randomly allotted to three subgroups; in one group ovulation was induced by the GnRH analog buserelin, in another by hCG, whereas a third group remained untreated. The synchronized groups were fixed-time inseminated; the untreated group bred to observed oestrus. Relative to untreated herd mates, pregnancy rate in cows subjected to a presynch protocol with buserelin as ovulation-inducing agent was 74%; for hCG it was 60%. In cows subjected to an ovsynch protocol, the corresponding relative pregnancy rates reached 138% in the case of buserelin and 95% in the case of hCG. Average service interval was shortened by 1 week in the presynch and delayed by 2 weeks in the ovsynch group. It may be concluded that fixed-time insemination of cows synchronized via ovsynch protocol with buserelin as ovulation-inducing agent is practicable and may help improve efficiency and reduce the work load involved with herd management in medium-sized dairy operations. The substitution of hCG for buserelin was found to be not advisable.

Subclinical endometritis has a major effect on the reproductive performance of dairy cows and also on the success of embryo collection. Thus it is important to minimize the number of false-negative diagnoses. In order to evaluate the question of whether or not a single cytobrush sample is representative of the whole endometrium, 53 German Holstein embryo donor cows in the northwest of Germany were examined via the cytobrush method at three different localizations of the uterus: the uterine body about 0.5 cm cranial of the cervical canal and both uterine horns about 1.5 cm cranial of the bifurcation. Although the mean percentage of polymorphonuclear neutrophils at the three locations is not significantly different (p = 0.64), the individual variations lead to the conclusion that more than one sample of the endometrium should be taken into account when diagnosing subclinical endometritis in embryo donor cows.

After the pioneering work of Rostand (1946 CR Acad. Sci. III 222, 1524–1525) and Polge et al. (1949 Nature 164, 666–669) on semen cryopreservation and the reports on the first live young born after storage of mouse embryos for up to 8 days (Whittingham et al. 1972 Science 178, 411–414) or 8 months (Whittingham and Whitten 1974 J. Reprod. Fertil. 36, 433–435) in liquid nitrogen, the world’s first calf was obtained in Cambridge nearly 40 years ago, after freezing and thawing of a bovine embryo (Wilmut and Rowson 1973 Vet. Rec. 92, 686–690). Since then, especially in humans (case reports), the birth of healthy offspring after long-term storage of semen, zygotes, and embryos has been repeatedly reported (e.g. Dowling-Lacey et al. 2011 Fertil. Steril. 95, 1120.e1–3). But practical experiences in different other species such as mice or cattle show contradictory results on the fertility after transfer of long-term-stored gametes or embryos. Here, we report the birth of normal Holstein calves after long-term embryo preservation. After storage for 15 to 22 years, 4 embryos were thawed (air-thaw for 10 s and 25°C water bath for 20 s), which had been originally frozen in 10% glycerol (3 embryos) or in 1.5 M ethylenglycol (1 embryo) following a slow freezing protocol. Removal of glycerol was accomplished with a 4-step dilution procedure and 10.3% sucrose (EMCARETM Thawing System, ICPbio Reproduction, Glenfield, New Zealand), whereas ethylenglycol was removed with a washing step in EMCARETM (Holding Solution, ICPbio Reproduction). The embryos were transferred into the uterine horn ipsilateral to the corpus luteum of recipient cows at 7 days post-oestrus. One embryo was classified as expanded blastocyst grade 1 (7-1), 2 embryos were classified as morulae grade 1 (4-1), and the embryo frozen with ethyleneglycol was classified as a morula grade 2 (4-2) (IETS classification; 40× magnification). Three pregnancies were established (1 × 7-1; 1 × 4-1, and 1 × 4-2), and 3 healthy bull calves were born (18.1.2011, stored 15 years; 6.3.2012, stored 22 years; and 29.3.2012, stored 15 years). These case reports document that long-term-stored bovine embryos maintained developmental competence for up to 22 years of cryopreservation in liquid nitrogen. To our knowledge these cases represent the “oldest” cryopreserved bovine embryos resulting in healthy calves in Germany.

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