Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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16 t h International Congress on Animal Reproduction 178 Poster Abstracts P457 Microfluidic sorting of boar spermatozoa Holt, W 1 *, Satake, N 1 , Smith, GD 2 , Alhaider, A 3 , Takayama, S 4 , Watson, PF 3 1Institute of Zoology, Zoological Society of London, United Kingdom; 2Department of Obstetrics and Gynecology, University of Michigan, United States; 3 Veterinary Basic Sciences, Royal Veterinary College, United Kingdom; 4 Department of Biomedical Engineering, University of Michigan, United States Microfluidic sperm sorter devices, developed at the University of Michigan, are able to sort a highly motile subpopulation of human spermatozoa, where motile spermatozoa traverse the boundary between two laminar flowing streams (one containing spermatozoa and the other comprising culture media alone) and are collected at an outlet reservoir (1). The aim of the present study was to investigate the application of this technology to the physical separation of putative boar sperm subpopulations. We have previously shown statistically that bicarbonate exposure significantly stimulates the motility of a (boar-dependent) sperm subpopulation (2), while others remain unaffected; physical separation of these populations has never been achieved. Percoll washed spermatozoa from 3 boars were incubated (10 min at 38ºC) in Tyrode’s based medium without bicarbonate, then 15mM bicarbonate/CO 2 was added to the medium before loading into sperm-sorters (provided by Strex Inc., Japan); the sorters were then left for 30 minutes at 38°C. Two outlet populations were collected and 60μl samples of each were subsequently analysed using a CASA system (Hobson Sperm Tracker, Hobson Tracking Systems, Sheffield, UK). In a preliminary study, spermatozoa were stained with SYBR14 and Propidium Iodide before loading into sperm sorters; videomicrography of the sorting channel showed that >95% of spermatozoa in the ‘sorted’ channel were live (PI−ve), although they were highly diluted. The proportion of immotile spermatozoa was reduced from 45% in the unsorted channel to 26% in the sorted channel. The proportion of progressively motile spermatozoa also decreased upon sorting (from 45% to 22%), but there was a surprising increase in the proportion of hyperactivated spermatozoa (from 10% to 52%). These results demonstrate the feasibility of using the sperm sorters for the isolation of a boar sperm subpopulation. Given that progressive motility is required for the sorting process to occur, the prevalence of hyperactivated spermatozoa after sorting suggests that the sorting may induce the final stages of capacitation. P458 Time oriented A.I. in cattle with reduced number of sperm cells Kanitz, W. 1 *, Becker, F. 1 , Bhojwani, S. 1 , Alm, H. 1 , Nehring, H. 2 , Nürnberg, G. 1 1Research Institute for the Biology of Farm Animals, Dummerstorf, Germany, 2Institute for Reproduction of Farm Animals, Schönow, Germany Among others number of spermatozoa per A.I. and time of insemination with regard to oestrus symptoms or ovulation are important determinants for pregnancy rate in cattle. In addition, the necessary number of spermatozoa per A.I. is under investigation with an aim of better utilisation of A.I. sires and the insemination of sorted spermatozoa. Because, data on fertilisation rate after A.I. with reduced sperm number are unavailable, we performed the following experiment. Altogether 116 heifers (German Holstein) received PGF 2 (0.5 mg Cloprostenol ® , Jenapharm, Germany; i.m.) between day 8 to 14 of oestrous cycle. 60 hours later 50 µg GnRH agonist (Depherelin ® ; Veyx, Germany, i.m.) was injected. Subsequently, 13 hours later the animals were inseminated once with frozen/thawed semen of 4 proven sires. The number of spermatozoa was 15x10 6 , 5x10 6 or 1x10 6 per A.I. (G1/G2/G3). After surgical recovery of the oviduct and the tip of the uterine horn ipsilateral to the C.l. embryos and oocytes were flushed from the oviduct on day 4 after insemination. The recovered oocytes and embryos were classified according their morphology and fixed using BFS medium (buffered formol solution, Merck, Germany). All oocytes and embryos were stained with HOECHST 33258 (Sigma, Germany) to identify accessory sperm cells. For statistical analyses fertilisation rates and portions of intact embryos were compared by means of Chi-squaretest. The influence of the factors sire, and sperm number on fertilisation rate was tested by GLM-procedure of SAS ® (Release 8.2, SAS Institute, Inc., Cary, NC 1999). Of the 116 heifers treated 106 animals ovulated (ovulation rate 91.5 %). The mean recovery rate for oocytes and embryos was 80.2 %. Mean fertilisation rates and the portions of intact embryos were 92.3/84.6 % (G1), 96.2/80.7% (G2) and 78.8/75.8 % (G3). Mean number of accessory sperm cells per embryo was significantly higher in G1 and G2 than in G3 (G1: 26.6±8.4, G2: 45.3±8.6, G3: 6.5±7.2). From the data obtained it can be concluded that time oriented A.I. can result in high fertilisation rate. Moreover, the portion of intact embryos does not depend on sperm number per insemination after time oriented A.I. Finally, the number of accessory sperm cells is not correlated with the portion of intact embryos after time oriented A.I. P459 A simple technique for minimal dose DIUI in PMSG multiple-ovulating dairy heifers Kornmatitsuk, B 1 *; Charoenyongyoo, P 1 ; Pattharanukulkit, K 1 ; Akkhawattanangkul, Y 1 ; Chaiprasat, S 2 ; Kornmatitsuk, S 1 1Faculty of Veterinary Science, Mahidol University, Phutthamonthon, Nakhon Pathom, 73170; 2 Livestock Semen Production Center-Inthanont Royal Project, Department of Livestock Development, Ministry of Agriculture and Cooperatives, Maung, Chiang Mai, 50300 The aim of the present study was to investigate an accomplishment of minimal dose deep intra-uterine insemination (DIUI) using a simple embryo transfer (ET) pistolet, in connection to relative fertilisation rates and early embryo qualities on day 7 post-service, studying in pregnant mare serum gonadotropin (PMSG) multiple-ovulating dairy heifers. Ten heifers of crossbred Holstein Frisian (≥75%) were included. They were characterised for their oestrous cycles, afterwards subjected to the superovulatory programme using PMSG/ Prostaglandin F2alpha (PGF2alpha) protocol, and fixed-time artificial insemination: Group 1, 3 heifers inseminated with 20×10 6 sperm cells at body of uterus; Group 2, 4 heifers inseminated with 10×10 6 sperm cells at tip of the uterine horns and Group 3, 3 heifers inseminated with 10×10 6 sperm cells at body of the uterus.. Embryos were nonsurgically recovered and categorised on 7 days subsequent to the insemination. On the day of each treatment, the ovaries of the heifers were ultrasonographically examined. Prior to the treatment, majorities of the follicles observed in all heifers were of small sizes (
16 t h International Congress on Animal Reproduction Poster Abstracts 179 P460 Improved sperm tail viability differentiation by coloured mounting medium Kovács, A 1-2* , Sarlós, P 1 , Egerszegi, I 1 , Oláh, J 2 , Révay, T 1 , Vass, N 2 and Jávor, A 2 1Research Institute for Animal Breeding and Nutrition; 2 Debrecen University, Centre for Agricultural Sciences, Institute for Animal Breeding Sciences Introduction Head membrane permeability and acrosome status of spermatozoa of different domestic mammals can be well differenciated by the combined trypan blue – Giemsa staining (Kovács A., Foote R.H.: Viability and acrosome staining of bull, boar and rabbit spermatozoa. Biotechnic & Histochemistry 67: 119-124, 1992; Nagy Sz. et al.: Evaluation of sperm tail membrane integrity by light microscopy. Theriogenology 52: 1153-1159, 1999), as well as by the similar Chicago sky blue - Giemsa (Kútvölgyi et al.: Viability and acrosome staining of stallion spermatozoa by Chicago sky blue and Giemsa. Biotechnic & Histochemistry 81: 109-117, 2006) staining. The evaluation of the tail membrane permeability may be often uncertain due to its narrowness and different backgrounds caused by seminal plasma and extender components. Materials and methods Both combined stainings are routinely applied in our laboratories using a yellow filter helping the better differentiation of the acrosome status. Preparations from fresh ram and boar, and frozen-thawed bull and ram semen samples were mounted with immersion oil (Merck 4699) control evaluated with a yellow filter, or with the same immersion oil saturated with Sudan I. (Peking Chemical Works, Peking, China) or with dimethyl yellow (Sigma D6760). The saturated solutions were the supernatants of the centrifugated oversaturated ones. Results and discussion Sudan I. or Dimethyl yellow solved in immersion oil resulted in not only a good differentiation of the acrosome status similarly to the control, but also a better distinguishing of membrane permeable „dead” and impermeable „live” tail domains of spermatozoa even in the case of background caused by the extender. The effect can be explained by the relief-like nature of the smears - the yellow mounting medium is more thick around the cells than above them. The yellow surroundings of the cells are lighter than the coloured „dead”, but darker than the unstained „live” sperm tails ensuring their easy and clear differentiation. The tail membrane of spermatozoa is more sensitive to the harmful effects of the freezing-thawing procedure than their head membrane, therefore its correct evaluation is extremely important for the quality control. P461 First foal born in Italy using flow cytometrically sorted spermatozoa Mari, G 1 *; Rizzato, G 1 ; Iacono, E 1 ; Merlo, B 1 ; Seren, E 2 ; Tamanini, C 2 ; Galeati, G 2 ; Spinaci, M 2 1 Veterinary Clinical Department, University of Bologna, Italy; 2 Dep. of Veterinary Morphophysiology and Animal Production, University of Bologna, Italy Introduction Sex-preselection of stallion spermatozoa is possible using a modified flow cytometer to separate spermatozoa into X and Y-chromosome bearing cells on the basis of DNA content. The objectives of this study were to evaluate vitality, motility characteristics and fertilizing ability of equine sexed semen. Methods Semen from 4 thoroughbred stallions of proven fertility (65% pregnancy rate per cycle) was diluted 1:1 with KMT and analysed by Computer Aided Sperm Analysis (CASA). Total motility (TM), progressive motility (PM), velocity average path (VAP) and rapid spermatozoa (RAP) were recorded. For sorting, samples were diluted to 200 x10 6 spermatozoa/ml and stained with Hoechst 33342 for 1.30 h at 35° C in the dark. Just prior to sorting a final concentration of 100 x10 6 spermatozoa/ml was reached and red food dye was added. A MoFlo SX® sperm sorter was used. Sorted spermatozoa were evaluated for motility parameters by CASA and for viability by SYBR-PI staining. The fertility of sexed spermatozoa was assessed by inseminating 4 mares (7 estrous cycles). Mares were histeroscopically inseminated at the utero-tubal junction, ipsilateral to the preovulatory follicle, with 5 x10 6 spermatozoa in 250 μl, 30-32 h after 2500 IU hCG administration for induction of ovulation. Pregnancy diagnosis was carried out 15 days after ovulation. Results Sperm motility characteristics were negatively affected by sorting (pre vs. post-sorting: TM 79.6±5.5 vs. 38.6±14.5; PM 32.3±9.4 vs. 10.6±5.4; VAP 116.4±12.4 vs. 99.9±13.3; RAP 67.0±12.0 vs. 30.2±12.0; p0.05, T student test). Pregnancy rate was 28.6% (2/7), and a pregnancy was loss at 30 days while the other was carried to term with the birth of a healthy filly. Conclusions Sperm motility parameters are negatively affected by the sorting process. On the other hand, viability is only slightly and nonsignificantly affected. Usually, mares are inseminated with 500 x 10 6 motile spermatozoa; being that the number of sexed spermatozoa/h is about 15 x 10 6 , and it would take too time to obtain the optimal dose, the histeroscopic insemination allows to use low numbers of sexed spermatozoa. Pregnancy rate obtained is lower than that reported in literature (Lindsey et al., 2002: 38% with 5 x10 6 motile spermatozoa), likely due to the lower number of motile spermatozoa in the inseminating dose used in the present study. The Authors wish to thank “Società Italiana Produttori Sementi”. P462 Viability of equine spermatozoa recovered from the epididymis cauda submitted to refrigeration Martins, MIM*; Nagao, JF; Gomes, RG Department of Veterinary Clinics, University of Londrina State (UEL), Brazil The recovery of spermatozoa from the epididymis cauda may be an important tool in equine reproduction because it makes possible the recovery of viable cells after the death of valuable stallions. The purpose of this study was to compare the viability of the spermatozoa recovered from the epididymis cauda submitted to refrigeration. Epididymals were obtained from five horses (English Thoroughbreds and two mixed –breed) from a slaughterhouse. During the transportation to the lab, the specimens were maintained in a 0,9% saline solution in an ice box. The recovery of the spermatozoa was accomplished by compressing the epididymis cauda and part of the deferent duct with help of an anatomic nipper on a Petri dish containing the extender Botu-Sêmen® (Biotech Ltda, Botucatu, Brazil). The sperm samples were evaluated for motility, vigor, sperm concentration, percentage of viable cells and morphology. The semen was diluted (final concentration of 100x10 6 /mL) and stored in 1,5 mL plastic tubes and packed in a transportation system Botu-Tainer® (Biotech Ltda, Botucatu, Brazil) for 18 hours. After this period, the tubes were transferred to a container with 400mL of water and kept in the refrigerator (4ºC) for 24 hours. The semen samples were analysed in three different moments: collection (M1), removal from the transportation boxes (M2) and after 24 hours of refrigeration at 4ºC (M3). The average motility (%), vigor (0-5), alive (%) and normal spermatic cells (%) were 64.0, 3.7, 90.6 e 60.0 (M1); 56.0, 2.9, 91.8 and 57.8 (M2); 56.0, 3.1, 88.4 and 56.0 (M3). The most frequent spermatic alterations were: abaxial tail insertion (6.3%), proximal cytoplasmic droplet (7.3%), distal cytoplasmic droplet (3.0%) and strongly bended tail (13.4%); probably because the spermatozoas were from the epididymis cauda. Transportation was considered efficient for the maintenance of spermatic quality after 24 hours at 4 ºC and no significant difference was observed among the evaluations. The process of spermatozoa recovery and refrigeration obtained from the epididymis cauda is promising, however, new experiments must be carried out aiming the analyses of in vivo and/or in vitro fertility of these cells.
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Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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