Hasan Ülker

CpG motif-based adjuvant as a replacement for Freund's complete adjuvant in a recombinant LHRH vaccine

This study compared: (1) Freunds complete adjuvant and CpG oligodeoxynucleotide (ODN) 2006 in water-in-oil emulsion as adjuvants; and (2) increasing doses of a recombinant ovalbumin-LHRH (ova-LHRH) fusion protein as an antigen for a contraceptive vaccine. Treatment groups (n=8 heifers/group) were: one untreated control group; five groups receiving CpG ODN with different doses of ova-LHRH (1.5; 2.3; 3.4; 5.1; and 7.6 mg); and one group receiving 3.4 mg ova-LHRH in Freunds. Heifers were immunized at weeks 0 and 14. All vaccine treatments caused gonadal regression and estrus suppression. CpG ODN is a suitable replacement for Freunds for LHRH immunization.

LHRH Fusion Protein Immunization Alters Testicular Development, Ultrasonographic and Histological Appearance of Ram Testis

This study was designed to evaluate the effectiveness of a luteinizing hormone releasing hormone (LHRH) fusion protein immunization on reproductive traits in ram lambs including the changes in histology and ultrasonographic appearance of testis. Thirty native ram lambs at 19 weeks of age were divided into control (C, n = 10), immunization (I, n = 10) and castration (E, n = 10) groups. Animals in immunization group were immunized against LHRH using ovalbumin-LHRH-7 (OL) protein generated by recombinant DNA technology as a primary and a booster injection at 19 and 23 weeks of age respectively. Animals were bled via jugular venepuncture at 2-week intervals to determine LHRH antibody and testosterone concentrations. Bi-weekly ultrasonographic examination of the testes was performed to determine the changes in ultrasonographic appearance as the age increased. Biopsied testicular tissues taken at 19, 29 and 41 weeks age were also evaluated. At 41 weeks of age, animals were slaughtered. Semen and epididymis were evaluated for the presence of sperm cells. Testicular development and sperm production were suppressed in the immunized animals. Semineferous tubule diameters decreased, basal membrane of the tubule was thickened and hyalinized in immunized ram lambs. While testes of control animals gained their normal ultrasonographic appearance as the age increased, immunized animals had uniform hypoechogenic testicular structure as observed at 19 weeks of age until slaughter. Simultaneous histological and ultrasonographic evaluations indicated that the changes in testicular histology could partly be monitored via ultrasonographic imaging. Nevertheless, it is difficult to claim that ultrasonographic image reflects the exact changes in such instances. In conclusion, these results indicate that recombinant OL fusion protein is effective in immunocastration in ram lambs and has a potential to be used as an alternative to physical castration. Further research studies should be conducted to help assess reproductive status of testes from ultrasound images.

The effect of immunization against LHRH on body growth and carcass characteristics in Karakas ram lambs

The effects of immunization against luteinizing hormone releasing hormone (LHRH) on body growth and carcass char- acteristics in Karakas ram lambs were investigated using recombinant LHRH fusion proteins. Recombinant fusion proteins, ovalbumin-LHRH-7 and thioredoxin-LHRH-7, were produced using recombinant DNA technology and expressed inE. coli. The control group (C, n = 5) was injected with ovalbumin and thioredoxin recombinant protein mixture, the immunization group (I, n = 6) was injected with a ovalbumin-LHRH-7 and thioredoxin-LHRH-7 recombinant fusion protein mixture (anti-LHRH vaccine) and, ram lambs in the elastrator group (E, n = 5) were castrated using elastrator bands. Animals in each group were weaned at 17 weeks of age and injected (primary immunization) with either mixture at 18 weeks of age or castrated. The C and I groups received a booster immunization 8 weeks later (26 weeks of age). Animals were housed in groups, weighed every 2 weeks and slaughtered at 36 weeks of age. Carcass of slaughtered lambs were chilled for 24 h at +4 ◦ C and evaluated for carcass characteristics. Immunization did not reduce growth rate and live body weights. Immunization and castration had no effect on carcass measurements and loin eye muscle area, hot and cold carcass weights, dressing percentage and wholesale cuts. Immunization against LHRH reduced testis weight and immunized animals had leaner carcasses than castrates. It was concluded that immunizing ram lambs against LHRH using new recombinant LHRH fusion proteins could be an alternative to physical castration to improve carcass quality in ram lamb production; however, further research is required to determine the effective immunization and slaughter age to improve carcass traits. Published by Elsevier Science B.V.

The effects of immunological castration against GnRH with recombinant OL protein (Ovalbumin-LHRH-7) on carcass and meat quality characteristics, histological appearance of testes and pituitary gland in Kıvırcık male lambs.

The aims of the study were to investigate the effects of immunization against GnRH using OL protein (Ovalbumin-LHRH-7) on feedlot performance, carcass, meat quality and some reproductive traits in Kıvırcık ram lambs. Ram lambs in the immunization (I, n=7) group were immunized against GnRH using OL protein and boosted 2 weeks later. Control (C, n=7) group was not treated. The animals were kept at pasture for 6 weeks after the first immunization, subjected to a 70 day fattening program, and then slaughtered. Growth performance, various carcass and meat quality characteristics were not affected from the immunization. GnRH immunization induced GnRH antibody production, suppressed testosterone production and testicular growth (P<0.01). Testicular structure was negatively affected from the immunization, but not pituitary. These results suggest that immunization against GnRH with OL could be an alternative castration technique in ram lambs without negatively affecting carcass and meat quality characteristics.

Changes in testicular development, ultrasonographic and histological appearance of the testis in buck kids immunized against LHRH using recombinant LHRH fusion protein.

This study was designed to evaluate the effectiveness of recombinant Ovalbumin-LHRL (OL) immunization on changes in testicular size, histological appearance and testosterone production in buck kids. Thirty native buck kids at 18 weeks of age were divided into three groups, control (n = 10), immunization (n = 10) and castration (n = 10) groups. Immunized animals received OL protein generated by recombinant DNA technology. Ultrasonographic and histological examinations of the testes were performed. Animals were slaughtered at 44 weeks of age. Semen and epididymides were evaluated for the presence of sperm cells. Immunized animals generated anti-LHRH antibodies. Testosterone production, testicular and accessory glands development and sperm production were suppressed in the immunized animals (p < 0.01). Semineferous tubule diameters decreased (p < 0.01), basal membrane of the tubule was thickened and hyalinized in immunized kids. Immunization affected ultrasonographic appearance of the testes drastically. While testes of control animals gained their normal ultrasonographic appearance as the age increased, immunized animals had uniform hypoechogenic testicular structure as observed at 18 weeks of age until slaughter. Simultaneous histological and ultrasonographic evaluations indicated that the changes in testicular histology could partly be monitored via ultrasonographic imaging; nevertheless, it is difficult to claim that ultrasonographic image reflects the exact changes in such instances. In conclusion, these results indicate that recombinant OL fusion protein is effective in immunocastration in buck kids and has a potential to be used as an alternative to physical castration. Further researches should be conducted to help assessing reproductive status of testes from ultrasound images.

GnRH or eCG treatment fails to restore reproductive function in GnRH immunized ewes

This study was designed to evaluate the potential of using eCG or GnRH in restoring reproductive functions in GnRH immunized ewes. Thirty-three multiparous Kivircik ewes were randomly assigned into either control group (n=11) or immunization group (n=22). Ewes were immunized against GnRH by injecting with a cocktail of ovalbumin-LHRH-7 (ovalbumin-GnRH-7) and thioredoxin-LHRH-7 (thioredoxin-GnRH-7) fusion proteins generated by recombinant DNA technology in April. 500 IU eCG or 0.008 mg GnRH analogue was used to induce ovulations. Serum GnRH antibodies were present in animals of the immunized group beginning the second week after the first immunization and maintained throughout the study (14 months). Immunization caused anestrus in immunized ewes. eCG or GnRH analogue administration given after 14 days progestagen (20 mg fluorogestone acetate, FGA) treatment during breeding season (mid July) did not induce ovulation in these ewes. Two more attempts with single or multiple eCG injections failed to induce ovulation in this group as well. It appears that the gonadotropin stimulation was not of adequate time since neither eCG nor GnRH administration was able to restore reproductive function in immunized animals. The immunization effect lasted more than a year. These results suggest that GnRH immunization exerts its effect via the hypothalamo-pituitary axis and that more than such stimulation is required to overcome the reproductive suppression.

Feedlot performance and carcass characteristics of ram lambs immunized against recombinant LHRH fusion proteins at 10 weeks of age

Body growth, feedlot performance and carcass characteristics of ram lambs (n = 16) immunized against luteinizing hormone-releasing hormone (LHRH) at 10 weeks of age with recombinant LHRH fusion proteins were investigated. Recombinant fusion proteins, ovalbumin–LHRH-7 and thioredoxin–LHRH-7 were produced using recombinant DNA technology. Animals were immunized (n = 8) against LHRH with ovalbumin–LHRH-7 and thioredoxin–LHRH-7 recombinant protein mixture in the Freund’s complete adjuvant. The immunization group received two booster injections 4 and 8 weeks later, with Freund’s incomplete adjuvant. Animals in control group (n = 8) were not treated. Animals were kept at relatively poor pasture until 27 weeks of age. This was followed by a 70 days finishing period that involved housing in groups and ad libitum feeding with concentrate. Carcasses were evaluated after chilling for 24 h at +4 ◦ C. Live weights, finishing weight, weight gain and average daily weight gain were similar between groups (P > 0.05). Carcass measurements, loin eye muscle area and back fat thickness were not affected from immunization. Immunization did not affect hot and cold carcass weights, dressing percentage, offal items and wholesale cuts weights. Immunized animals had smaller testis, chop and bone weights than control animals (P < 0.05). It was concluded that immunological castration could be achieved at 10 weeks of age in ram lambs using new recombinant LHRH fusion proteins and used in finishing programs without adverse effect on growth rate, feedlot performance and carcass characteristics. Nevertheless, the effectiveness of these proteins should be further evaluated with more animals.

The Effectiveness of recombinant OL fusion protein (ovalbumin-LHRH-7) in suppressing reproductive functions when injected in single-dose vaccination protocols with different adjuvants

The objective of this study was to evaluate the effectiveness of recombinant LHRH fusion protein, Ovalbumin-LHRH-7 (OL), using a single-dose vaccination protocol in combination with different adjuvants in suppressing reproductive functions in buck kids. For this purpose, either a mixture of free OL antigen and encapsulated OL antigen, or encapsulated OL antigen was used. Thirty-nine native buck kids at 12 weeks of age were divided into control (n=7) and treatment groups (n=8 bucks/group). The four treatment groups were formed according to the different vaccine formulations: Group CpG received 0.5mg free OL protein together with 1.0mg of encapsulated protein with CpG adjuvant. Group mFCA received 0.5mg free OL protein together with 1.0mg of encapsulated protein with modified Freunds complete adjuvant. Group IS received 1.5mg encapsulated OL protein with a mix of inulin and saponin adjuvants. Group ISmFCA received 1.5mg encapsulated OL protein with a mix of inulin, saponin and modified Freunds complete adjuvants. Scrotal circumference in CpG and mFCA groups were significantly smaller than that of Control, IS and ISmFCA groups (P<0.05). Numbers and percentage of bucks having spermatozoa in their ejaculate were significantly lower in CpG and mFCA groups (P<0.05). OL immunization completely suppressed sperm production, except one buck, in CpG and mFCA groups (P<0.05). These results imply that it is possible to use OL protein in a single injection protocol for the purpose of immunocastration. Further investigation with a larger number of animals should be carried out to determine the longevity of response to a single injection.

Hayvancılık Destekleme Politikalarına Çiftçilerin Yaklaşımlarının Bölgelerarası Karşılaştırmalı Analizi / A Regional Comparative Analysis of Farmers’ Approaches to Livestock Support Policies

OZET : Spermatogonial transplantasyon, verici bir hayvandan izole edilen spermatogonial hucrelerin bir dizi farkli yontemler kullanilarak testisleri germ hucrelerinden temizlenip infertil hale getirilmis olan alici hayvanin testislerine transfer edilmesi islemidir. Aktarilan spermatogonial hucreler kendilerini seminefer tuplerin bazal tabakasina konuslandirirlar ve spermatogenesisi baslatarak germ hucreleri tamamen bosaltilan alici hayvanin testisini yeni germ hucreleri ile doldurarak fertil bir testis meydana getirirler. Bu teknige iliskin calismalar spermatogonial kok hucrelerinin verici hayvanlardan izole edilmesi, kultur edilmesi, dondurularak saklanmasi, transplantasyon yapilmadan once alici testislerdeki spermatogonianin yok edilerek temizlenmesi, aktarilan hucrelerin alici testislerde varliginin belirlenmesi icin marker sistemlerinin gelistirilmesi ve spermatogonial transplantasyonun farkli turler arasinda uygulanisi seklinde siralanabilir. Bu teknigin ciftlik hayvanlarinda damizlik degeri belirleme zamanini azaltma, normal genetik degere sahip alici hayvanlari kisa bir sure icerisinde vericinin genetik degerine sahip germ hucresi ureten hayvanlara donusturebilme, transgenik ciftlik hayvanlari uretme gibi amaclarla yaygin olarak kullanilabilecegi ongorulmektedir. Spermatogonial transplantasyon teknigi spermatogenesis, spermatogonial kok hucreleri ve gamet-testis fonksiyonlari ve interaksiyonlarini arastirmada oldukca kullanisli bir yontemdir. Bu teknigin temel bilimden klinik uygulamalara, nesli tukenen turlerden hayvan islahina bircok alanda yararli uygulamalara yon verecegi ongorulmektedir. Anahtar kelimeler: Spermatogonial transplantasyon, testis, hayvan islahi Spermatogonial Transplantation and Its Utilization in Animal Breeding ABSTRACT : Spermatogonial transplantation is a technique which spermatogonial germ cells isolated from a donor animal are transplanted into the testes of recipient animal whose testes were depleted from germ cells. Transferred spermatogonial germ cells translocate themselves to the base of semineferous tubules, repopulate the germ-cell depleted testes of recipient animal by reinitiating spermatogenesis and restore the fertility. Isolation, culture and cryopreservation of spermatogonial stem cells from donor animals, depleting the germ cells of recipient testes prior to transplantation, developing marker systems to determine donor derived spermatogonia in the recipient testes and interspecies transplantation could be considered as the main topics of spermatogonial transplantation studies carried out so far. This technique is speculated to be used for various purposes such as decreasing the time needed to prove the genetic value of the livestock animals, converting the genetically average recipient animals into genetically superior germ cell producing animals in a short time and generating transgenic livestock. Spermatogonial transplantation is a useful technique to be used in investigating spermatogenesis, spermatogonial stem cells, gamete-testis functions and interactions. It is thought that this technique can be utilized in a variety of the areas such as basic science, clinical applications, endangered species and animal breeding. Key words: Spermatogonial transplantation, testis, animal breeding