Pregnancy-associated glycoproteins (PAG) in buffalo species (Bubalus bubalis): Purification and pregnancy fullow-up Olimpia Barbato Dip. Scienze Biopatologiche ed Igiene delle Produzioni Animali ed Alimentari Sez. Fisiologia Veterinaria Facoltà di Medicina Veterinaria di Perugia (Italia)

WORLD’s BUFFALO World Buffalo population: 180 million (FAO 2010) Italian Buffalo population: 400.000 The world’s buffaloes are classified in two groups: the African and the Asian. The two groups have been assigned different generic names: Syncerus and Bubalus. African and Asian buffaloes are from different lineages, separated from the other bovines during the Pleistocene (

African Asian BUFFALO Syncerus caffer Bubalus bubalis Type Swamp Type River India - Pakistan Middle est RUSSIA EUROPE Southeats Asia Cina – Australia Philippines The world’s buffaloes are classified in two groups: the African and the Asian. The two groups have been assigned different generic names: Syncerus and Bubalus. African and Asian buffaloes are from different lineages, separated from the other bovines during the Pleistocene ( Italian Mediterranean Race

The FIRST purification… 14 weeks The world’s buffaloes are classified in two .

The FIRST purification… These sequences revealed the existence of three PAG never described in other species Sequences: * Mid-Pregnancy ** Late-pregnancy

The FIRST purification… 1. Confirmed both molecular mass and amino acid N-terminal sequence multiplicities within the buffalo PAG family 2. Revealed the existence of THREE PAG NEVER FOUND IN OTHER SPECIES 3. Confirmed the efficacy of VVA chromatography in enrichment of placental glycoproteins expressed in ruminant species In conclusions, our results confirm both molecular mass and amino acid N-terminal sequence multiplicities within the buffalo PAG family; Revealed the existence of THREE PAG NEVER DESCRIBED IN OTHER SPECIES; 3.Confirm the suitability of VVA chromatography in enrichment of placental glycoproteins expressed in ruminant species

The SECOND purification… Single placentome a:caruncles b: cotyledons The world’s buffaloes are classified in two groups: the African and the Asian. The two groups have been assigned different generic names: Syncerus and Bubalus. African and Asian buffaloes are from different lineages, separated from the other bovines during the Pleistocene (Pregnancy-associated glycoproteins make up a large family of glycoproteins specifically expressed by the outer epithelial cell layer of the placenta in several Eutherian species. They are synthesized by the mono and binucleate trophoblastic cells, some of them being secreted in maternal blood from the moment when the conceptus becomes more closely attached to the uterine wall and the formation of placentomes begins. The world’s buffaloes are classified in two Pleistocene ( In the pregnant animals, PAGs are detectable in the maternal circulation, depending on species, from Day 21 after breeding till parturition.

The SECOND purification… The world’s buffaloes are classified in two groups: the African and the Asian. The two groups have been assigned different generic names: Syncerus and Bubalus. African and Asian buffaloes are from different lineages, separated from the other bovines during the Pleistocene (Pregnancy-associated glycoproteins make up a large family of glycoproteins specifically expressed by the outer epithelial cell layer of the placenta in several Eutherian species. They are synthesized by the mono and binucleate trophoblastic cells, some of them being secreted in maternal blood from the moment when the conceptus becomes more closely attached to the uterine wall and the formation of placentomes begins. The world’s buffaloes are classified in two Pleistocene ( In the pregnant animals, PAGs are detectable in the maternal circulation, depending on species, from Day 21 after breeding till parturition.

The SECOND purification… 1. Confirmed the multiplicity of PAG expression in buffalo placenta 2. Revealed the existence of ELEVEN wbPAGs NEVER FOUND IN OTHER SPECIES 3. A new RIA (RIA-860) was developed and used to distinguish between pregnant and non –pregnant water buffalo females In conclusions, our results confirm both molecular mass and amino acid N-terminal sequence multiplicities within the buffalo PAG family; Revealed the existence of THREE PAG NEVER DESCRIBED IN OTHER SPECIES; 3.Confirm the suitability of VVA chromatography in enrichment of placental glycoproteins expressed in ruminant species

The isolation of new antigens from buffalo placenta allowed the production of ANTIBODIES against buffalo PAGs. This production is useful in developing HOMOLOGOUS RIA technique currently used for pregnancy diagnosis and physiopathological investigations (…embryo mortality) in farm animals. The production of specific antisera can be very useful in immunohistochemical and immunocytochemical studies of PAG expression in fetomaternal interfaces

Plasma concentrations of PAG measured by using AS°#860 in non-pregnant and pregnant buffalo cows Day after AI Concentrations of PAG (ng/mL) Non-pregnant (n = 26) Pregnant (n = 33) Day 0 Mean PAG (ng/mL) 0.48 ± 0.04 0.41 ± 0.04a [min–max] [0-1.0] [0-0.8] Day 30 0.46 ± 0.06* 2.86 ± 0.39a,b,* [0.85-11.37] Day 37 0.27 ± 0.05** 11.60 ± 1.61b,** [3.12-49.00] The RIA-system was shown to be sensitive, linear, reproducible, accurate and specific in measuring PAG concentrations in buffalo plasma samples

PAG trend during pregnancy…

PAG trend during post partum… The PAG concentration disappears just 40 days after delivery

49 52 The world’s buffaloes are classified in two groups: the African and the Asian. The two groups have been assigned different generic names: Syncerus and Bubalus. African and Asian buffaloes are from different lineages, separated from the other bovines during the Pleistocene (Pregnancy-associated glycoproteins make up a large family of glycoproteins specifically expressed by the outer epithelial cell layer of the placenta in several Eutherian species. They are synthesized by the mono and binucleate trophoblastic cells, some of them being secreted in maternal blood from the moment when the conceptus becomes more closely attached to the uterine wall and the formation of placentomes begins. The world’s buffaloes are classified in two Pleistocene ( In the pregnant animals, PAGs are detectable in the maternal circulation, depending on species, from Day 21 after breeding till parturition.

ng/mL Day AI The world’s buffaloes are classified in two groups: the African and the Asian. The two groups have been assigned different generic names: Syncerus and Bubalus. African and Asian buffaloes are from different lineages, separated from the other bovines during the Pleistocene (Pregnancy-associated glycoproteins make up a large family of glycoproteins specifically expressed by the outer epithelial cell layer of the placenta in several Eutherian species. They are synthesized by the mono and binucleate trophoblastic cells, some of them being secreted in maternal blood from the moment when the conceptus becomes more closely attached to the uterine wall and the formation of placentomes begins. The world’s buffaloes are classified in two Pleistocene ( In the pregnant animals, PAGs are detectable in the maternal circulation, depending on species, from Day 21 after breeding till parturition. The PAG is detectable in buffalo cow from day 25 after breeding….until parturition

PAG profile in buffaloes with EM

PAG profile in buffaloes with EM Buffalo cow N° 1 Buffalo cow N° 2

Clinical application of PAG RIA for pregnancy follow-up… Measurement of PAG concentrations in buffalo cows maternal blood permits: Pregnancy diagnosis (…from 25 days after fecondation) and confirmation (reproduction management) Follow up the trophoblastic function (EM, EF, reproductive physiology, pathologic conditions affecting pregnancy) In practice, the measurement of PAG concentrations in peripheral maternal circulation has been used fort both pregnancy confirmation and the follow-up of the trophoblastic function. The first aspect can help veterinarians and breeders in the management of reproduction, while the second represents a powerful tool for investigators involved in studying factors affecting embryo and fetal mortality and embryo biotechnology.

ALSO IN BUFFALO: the concentrations of Pregnancy-Associated Glycoproteins can help… Farmers… - For pregnancy diagnosis in cows, sheep, goat and buffalo - alone or together with other sero diagnosis: brucellosis, leucosis, IBR, BVD... Veterinarians… - When developing rectal palpation or ultrasound investigations: - in order to confirm a diagnosis… The concentration of PAG can help FARMER for pregnancy diagnosis in cow, sheep, goats, buffalo, isolated or in conjunction with other sero diagnosis; VETERINARIANS when developing rectal palpation or ultrasound investigations; RESEARCHERS when studying embryonic or fetal mortality Researchers… - When studying embryonic or fetal mortality, the consequence of diseases, of nutrition…

Acknowledgements Team of Prof. J. F. Beckers: Physiology of Reproduction, Faculty of Veterinary Medicine, University of Liege, Belgium. Dott. Noelita Melo de Sousa: Physiology of Reproduction, Faculty of Veterinary Medicine, University of Liege, Belgium.

Thanks For Your Attention During gestation, several hormones and proteins are secreted by the maternal and fetal endocrine system. The dialogue between CL and conceptus is very important for a successful outcome of gestation. The hormones originating from the CL should influence the secretion of the endometrium and trophoblast molecules.