FREE FULL TEXT
VOLUME 9, NUMBER 2
ABSTRACTS
Factors and methods of pig oocyte and embryo quality improvement and their application in reproductive biotechnology
Barbara Gajda1
Department of Biotechnology of Animal Reproduction, National Research Institute of Animal Production, Balice/Krakow, Poland
Summary
Compared to other mammalian species, pig oocytes and embryos are characterized by high lipid contents stored mainly as lipid droplets in the cytoplasm.
This fact has a negative influence on manipulations on oocytes and embryos and, in general, biotechnological procedures are much less advanced in pigs than
in cows. This paper discusses current methods for modifying porcine oocytes and embryos using in vitro culture or microsurgical manipulation, chemical agents
such as cytochalasin B or D, physical means such as centrifugation or increased pressure and the biotechnological implications of these procedures. The
presented methods make it possible to modify the characteristics of oocytes and embryos and thus increase their susceptibility to cryopreservation and cloning.
Reproductive Biology 2009 9 (2): 97-112
1Corresponding author: Department of Biotechnology of Animal Reproduction, National Research Institute of Animal Production, Krakowska str. 1, 32-083 Balice/Krakow, Poland:
e-mail: bgajda@izoo.krakow.pl
Characteristics of spermatozoa of whole ejaculate and sperm-rich fraction of dog semen following exposure to media varying in osmolality
Rafal Strzezek1, Leyland Fraser
Department of Animal Biochemistry and Biotechnology, University of Warmia and Mazury, Olsztyn, Poland
Summary
This study aimed to analyze the effects of different osmolalities on the characteristics of spermatozoa originating from whole ejaculates
(WE; including the prostatic fluid) and the sperm-rich fractions (SRF). Ejaculates, collected from four mixed-breed dogs, were exposed for 10 min
at room temperature to Tris-fructose-citrate (TFC) solution with osmolality ranging from 150 to 1100 mOsm. After treatment spermatozoa
were evaluated by microscopic analysis of motility and fluorescent assessments of plasma membrane integrity (carboxyfluorescein diacetate and
propidium iodide, CFDA/PI) and mitochondrial function (rhodamine 123, R123). Irrespective of the sperm source, there was a complete loss of
motility when spermatozoa were exposed to TFC solution with 1100 mOsm. There were no marked differences in the sperm characteristics between
media with 300 and 350 mOsm, regardless of the ejaculate collection procedure. However, a marked reduction in motility of spermatozoa
retrieved either from the WE or SRF was observed after exposure to different anisosmotic conditions (150, 550 and 800 mOsm). In all dogs, spermatozoa
from the WE exhibited greater osmotolerance in terms of plasma membrane integrity and mitochondrial function when exposed to anisosmotic
conditions (150, 550, 800 and 1100 mOsm). There were inter-dog variations in response to various osmotic conditions. The findings of this
study indicated that spermatozoa from the WE tolerated exposure to a wider range of osmolality than those from the SRF. It seemed that the
presence of prostatic fluid of dog semen rendered the sperm membrane structures less susceptible to osmotic stress.
Reproductive Biology 2009 9 (2): 113-126
1Corresponding author: Department of Animal Biochemistry and Biotechnology, Faculty of Animal Bioengineering, University of Warmia and Mazury in Olsztyn, Oczapowskiego Street 5, 10-718 Olsztyn-Kortowo, Poland:
e-mail: rafi@uwm.edu.pl
Pregnancy-Associated Glycoprotein (PAG) family: transcripts and gene amplicons in camelids
Marta Majewska1,7, Grzegorz Panasiewicz1,3, Karl Klisch4, Louis V.M. Olivera5, Javier M. Mamani5, Mahmoud M. Abd-Elnaeim6, Bozena Szafranska2,3
3Department of Animal Physiology, University of Warmia and Mazury in Olsztyn, Poland;
4Neuroanatomy, Medical School Hannover, Hannover, Germany;
5National University of Altiplano, Puno, Peru;
6Department of Anatomy and Histology, Assuit University, Egypt;
7Nucleagena Ltd., Laboratory of Molecular Genetics, Warszawa, Poland
Summary
In this study, the placental localization of PAG-like transcripts and genomic existence of PAG-like amplicons in new-world
(Lp, Lama pacos, alpaca) and old-world camelids (Cb, Camelus bactrianus, bactrian; Cd, Camelus dromedarius; dromedary) are
reported for the first time. Sections of Lp (150-347 days post coitum), Cd (43-90 cm crown-rump length) and Cb (term) placentas
were used for heterologous (ht; cross-species) autoradiographic in situ hybridization (aISH) with single-stranded diagnostic
(antisense) or control (sense) [α-35S]dATP-labeled 323 nt porcine PAG8 (pPAG8) cDNA probes produced by asymmetric PCRs. The
aISH with antisense 35S-pPAG8 probe identified camelid PAG-like (LpPAG, CbPAG and CdPAG) mRNA expression restricted to chorionic
epithelium cells within placentas of camelids. In addition, genomic DNA (gDNA), isolated from placental sections were used as
templates for camelid PAG-like gene amplicon production by PCR. Specificity of the obtained multiple camelid gDNA PAG-like amplicons
was confirmed by double ht-Southern hybridizations with [α-32P]dATP-labeled 611 bp pPAG5 and pPAG10 double-stranded cDNA probes.
The double ht-Southern hybridizations of camelid gDNA amplicons (with pPAG5 and -10 probes) allowed the identification of length-polymorphism
of LpPAG, CbPAG and CdPAG genes, coding catalytically active and potentially inactive forms. Such an application of porcine PAG probes may be
advantageous for future identification of still undiscovered PAG-like families in other eutherian species.
Reproductive Biology 2009 9 (2): 127-150
1The first two authors contributed equally to the experimental work described in this paper.
2Corresponding author: Department of Animal Physiology, University of Warmia and Mazury in Olsztyn, 10-719 Olsztyn-Kortowo, Oczapowskiego Str 1A/222, Poland:
e-mail: szafran@uwm.edu.pl
Purification and partial characterization of proteinase inhibitors of equine seminal plasma
André Belico Vasconcelos1,2, Alexandre Martins Costa Santos3, Jamil Silvano Oliveira4, Monique de Albuquerque Lagares5, Marcelo Matos Santoro4
2Institute of Veterinary "José Caetano Borges", University of Uberaba,
3Department of Physiology Science Federal University of Espirito Santo,
4Department of Biochemistry Immunology,
5Department of Veterinary Clinic and Surgery Federal University of Minas Gerais, Brazil
Summary
The aims of the study were: 1/ to isolate and identify equine seminal plasma proteinase inhibitors,
2/ to evaluate their inhibitory potential, and 3/ to test a correlation between protein concentration in seminal plasma
supernatant (obtained after precipitation with 36% ammonium sulfate) and stallion sexual maturity. Seminal plasma proteins obtained from six
stallions were chromatographed in a Superose 12 (FPLC system) column followed by C18 HPLC reverse-phase. Inhibition of trypsin
amidase activity was evaluated in the collected fractions. Active proteins with a molecular mass of 6.3-7.0 KDa were
identified using mass spectrometry. The older stallions showed a reduction in total seminal plasma protein concentration, but had similar
concentrations of proteinase inhibitors (0.28±0.10 mg/ml) in seminal plasma supernatant. Different proteinase inhibitor isoforms were
found in semen of all stallions which suggests that the isoforms may be used as biomarkers of individual animals.
Reproductive Biology 2009 9 (2): 151-160
1Corresponding author: Institute of Veterinary "José Caetano Borges", Uberaba, Minas Gerais, Av. Tutunas 720, Postal code 38061-500, Brazil:
e-mail: andre_belico@yahoo.com.br
Local effect of progesterone infusion into the porcine ovarian artery on androgen and estrogen secretion during the middle luteal phase
Barbara Wasowska1, Stanislawa Stefanczyk-Krzymowska
The use of a hypo-osmotic swelling (HOS) test on sperm of the pig (Sus scrofa domesticus), emu (Dromaius novaehollandiae), Asian elephant (Elephas maximus), hamadryas baboon (Papio hamadryas hamadryas), and central rock rat (Zyzomys pedunculatus)
Phillip Matson1,2,3, Wendy Kappelle2, Irek Malecki4
The effects of prolactin and corticosterone on insulin binding to rat Leydig cells
Muthukumar Karthikeyan1,2, Jagadeesan Arunakaran3, Karundevi Balasubramanian3
FREE FULL TEXT
Department of Local Physiological Regulations, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland
Summary
The present study was undertaken to elucidate whether an increased, but physiological, amount of progesterone (P4) supplied to the porcine
corpus luteum (CL) affects luteal secretion of androgens and estrogens in freely moving gilts. On day 9 of the estrous cycle, the jugular veins
as well as both ovarian arteries and both ovarian veins of gilts were cannulated. Progesterone was infused into the right ovarian arteries of
experimental gilts (n=5) on days 10, 11 and 12 of the estrous cycle at a rate adequate to physiological retrograde transfer found during the
middle luteal phase of the cycle. The left ovarian arteries of the experimental gilts were infused with saline. Both ovarian arteries of the
control gilts (n=5) were infused with saline. The P4 infusion rate was 0.62 μg/min (10 day), 2x0.62 μg/min (11 day) and 3x0.62 μg/min (12 day).
Blood samples were collected from the jugular vein and both ovarian veins of the experimental and control gilts on days 10-12 of the estrous cycle
before and after P4 or saline infusion. The mean plasma androstenedione (A4) level in the ovarian vein ipsilateral to the P4-infused ovary was
higher (p<0.01) on days 10-12 of the estrous cycle than those found in the contralateral ovarian vein of the experimental gilts as well as the
control gilts. The ovarian venous level of testosterone (T) in the ovarian vein ipsilateral to the P4-infused ovary on days 10-12 of the estrous
cycle was not significantly different (p>0.05) from those found in the contralateral ovarian vein of the experimental gilts and ovarian vein of the
control gilts. However, on day 12, a decrease in T concentration was demonstrated in the ovarian vein ipsilateral to the P4-infused ovary in comparison
to those of the contralateral and control ovarian veins. The mean plasma 17β-estradiol (E2) level in the ovarian vein ipsilateral to the P4-infused
ovary was lower on days 10-12 than those found in the contralateral ovarian vein of the experimental gilts and in the ovarian vein of the control gilts
(p<0.001). The results of the present paper indicate that local elevation of P4 concentration in blood supplying the ovary during the middle luteal
phase of the porcine estrous cycle affected local secretion of androgens and estrogens. The effect of P4 on the secretion of androgens and estrogens
suggested the existence of a short regulatory loop of a positive feedback for A4 secretion and a negative feedback for E2 secretion.
Reproductive Biology 2009 9 (2): 161-179
1Corresponding author: Division of Reproductive Endocrinology and Pathophysiology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, 10-747 Olsztyn, Tuwima 10, Poland:
e-mail: bwasow@pan.olsztyn.pl
FREE FULL TEXT
2Perth Zoo, South Perth, Western Australia,
3School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, Western Australia,
4School of Biology, University of Western Australia, Crawley, Western Australia, Australia
Summary
A hypo-osmotic swelling test using TALP-HEPES medium over a range of 50 to 300 mOsm/kg was applied to sperm from domestic and endangered species.
Maximal responses of curling of the sperm tails were seen over a range of osmolalities for epididymal sperm from the pig (100 mOsm/kg), hamadryas
baboon (range 50-125 mOsm/kg), and central rock rat (range 50-100 mOsm/kg), and the ejaculated sperm from the emu (50 mOsm/kg) and the Asian elephant
(range 75-150 mOsm/kg). A solution of TALP-HEPES medium at 100 mOsm/kg would be suitable to obtain the maximal response in this range of mammals tested,
though it would need to be diluted to at least 50 mOsm/kg when testing the viability of the emu sperm.
Reproductive Biology 2009 9 (2): 181-187
1Corresponding author: Keogh Institute for Medical Research, Sir Charles Gairdner Hospital, Nedlands 6009, Australia:
e-mail: phillipmatson@optusnet.com.au
FREE FULL TEXT
2Reproductive Medicine Unit, Department of Obstetrics & Gynecology, Christian Medical College & Hospital;
3Department of Endocrinology, Dr ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Tamilnadu, India
Summary
The influence of prolactin (PRL) and corticosterone on insulin binding to purified rat Leydig cells was assessed in vitro. The lowest dose of
PRL (50 ng/ml) increased (p<0.05) and the remaining PRL concentrations (100, 150, 200, 250 ng/ml) decreased (p<0.05) the insulin binding to
Leydig cells. All doses of corticosterone (150, 200, 250, 300 ng/ml) except the lowest one (100 ng/ml) decreased the insulin binding. In
conclusion, hyperprolactinemia or excess glucocorticoids associated with an impairment of testicular steroidogenesis may be mediated by a
defective insulin binding to Leydig cells.
Reproductive Biology 2009 9 (2): 189-194
1Corresponding author: Reproductive Medicine Unit, Department of Obstetrics & Gynecology, Christian Medical College & Hospital, Vellore-4,Tamilnadu, India:
e-mail: mmuutthhuu@yahoo.com