Melamine excretion pathways in lactating dairy cows

Calitz, Tanja (2013-03)

Thesis (PhD)--Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: In this study, five trials were conducted to examine in vitro and in vivo degradation, excretion and absorption parameters of melamine (MEL) in dairy cows that have not been studied before or where limited information is available. The first two trials were in vitro studies conducted to determine the extent of MEL degradation in rumen liquor and the effects of MEL on ruminal ammonia (NH3) and volatile fatty acid (VFA) concentrations. For both trials, rumen liquor was collected from ruminally cannulated lactating Holstein cows. For the first and second trial, rumen liquor was collected from three and two cows, respectively. For both trials, Erlenmeyer flasks contained 1 g substrate and 100 mL incubation medium consisting of 20 mL rumen liquor and 80 mL reduced buffer solution. In the first trial, each flask contained 100 mg of MEL, resulting in an initial MEL concentration of 1000 mg/L. The flasks were incubated at 39° C for 0 (Control), 6, 24 or 48 hours under strictly anaerobic conditions. In all the trials, MEL concentrations were determined by LC/MSMS. MEL degradation was low after 6 and 24 h of incubation (3.2 and 5.5%, respectively) and increased to 13.6% after 48 h of incubation. In the second trial where VFA and NH3 concentrations were determined, the flasks contained either 0 (Control), 0.2 (T1) or 0.4 mg (T2) of MEL. The flasks were incubated for 6, 24 or 48 h. Treatment had no effect on individual or total VFA concentrations or NH3 concentrations at 6 and 48 h. At 24 h, T2 resulted in an inexplicable higher NH3 concentration. This study showed that the addition of melamine would not result increased rumen NH3 concentrations in vitro. Melamine would also not affect the production of different VFA’s. Therefore, it was concluded that the rumen micro-organisms present in rumen liquor would be unable to utilize MEL as a source of nitrogen and that the microbial production of VFA’s remains unaffected by the presence of MEL. In the third trial, MEL excretion in lactating cows was determined. Five cows were randomly allocated to treatments according to a 5 x 5 Latin square design. Cows received the treatment diets for 7 d followed by 8 d of MEL withdrawal during each of the five periods. The experimental treatments were formulated to provide a daily MEL intake of 0 (M0), 500 (M1), 1000 (M2), 5000 (M3) or 10000 mg (M4) via 15 kg of dairy concentrate pellets. Calculations based on the work of Newton & Utley (1978) suggested that a melamine intake of 0.16 g/kg of live weight would not result in detrimental health effects of ruminant animals. Therefore, a 600 kg lactating dairy cow should not be at risk when consuming 100 g of melamine. In this trial, the highest melamine treatment (M4 = 10 g/d) included a 10-fold safety factor from the suggested safe amount from the work of Newton & Utley (1978) and should not pose a health risk to the cows. Treatments had no effect on DMI, milk yield or milk composition. MEL was detected in the milk 8 h after initial MEL ingestion, increased rapidly and peaked on d 3 and was undetectable after 8 d. Treatments had no effect on MEL excretion efficiencies which ranged from 1.5 to 2.1%. The mean apparent digestibility of MEL was 78%. Mean faecal and urinary MEL excretions were 22 and 54 % of ingested MEL, respectively. Higher milk, urine and faecal MEL concentrations were observed with higher levels of dietary MEL. It was concluded that MEL appeared in the milk soon after first ingestion and a withdrawal period of 8 d was required for all milk, faecal and urine samples to reach undetectable levels of MEL. Urine and faeces were the primary routes for MEL excretion. The fourth trial was conducted to determine MEL absorption by the mammary gland in lactating dairy cows through arterio-venous (A-V) difference. Five cows received 10 g of MEL/d for three consecutive days. Day 3 of the trial was selected for commencement of blood sampling as previous studies (Cruywagen et al., 2009; Shen et al., 2010; Sun et al., 2011) reported the milk melamine concentration to reach a peak on d 3 of continuous melamine consumption by dairy cows. Early on d 3, catheters were inserted into the caudal superficial epigastric vein (milk vein) and caudal auricular artery. The blood sampling period commenced after residual milk removal from the udder following oxytocin administration. Blood from both locations were collected hourly for 9 hours. Following the final blood collection, oxytocin was administered again, catheters were carefully removed and cows were milked immediately thereafter. All blood samples were centrifuged and the decanted plasma was analysed for MEL, as well as for amino acid contents to calculate mammary blood flow. The positive MEL flux (calculated from A-V difference) confirmed net absorption of MEL into the mammary gland with an efficiency of absorption of 0.29%. Melamine excretion into milk was 5.63 mg/h. The mean plasma and milk MEL concentrations were 5.2 and 3.9 mg/kg, respectively. Melamine excretion efficiency to milk, expressed as percentage of the ingested amount, was 1.47%. It was concluded that melamine ingested by cows will result in net MEL absorption by the mammary gland, but that the absorption efficiency is low. The final trial of the study aimed to determine the effects that fermentation processes during the manufacturing of cheese, yoghurt and kefir would have on their MEL content if these products were made from MEL contaminated milk. Another objective was to determine if MEL in cheese would be degraded during the curing process. Cheese, yoghurt and kefir were made from milk with a MEL content of 6.77 mg/kg. The cheese was then cured for 2 wk at 6° C. The MEL contents of the yoghurt and kefir were 6.76 and 6.78 mg/kg, respectively, indicating that the different fermentation processes used in yoghurt and kefir production had no effect on their MEL content and that MEL was not degraded during the short fermentation periods. The percentage of milk MEL partitioned to whey and cheese were 97.4 and 6.5 %, respectively. It was concluded that the different fermentation processes involved during the manufacturing of yoghurt and kefir from MEL tainted milk did not decrease the MEL concentration. The milk MEL was predominantly partitioned to whey, with little MEL transferred to cheese. It was also concluded that MEL was not degraded in cheese during a 2-wk curing period. It was finally concluded that dietary MEL is readily absorbed by dairy cows and mainly excreted via the urine. The mammary gland has a low affinity for MEL absorption and approximately 2% of ingested MEL is excreted in the milk. When cheese is made from MEL tainted milk, the majority of MEL will concentrate in the whey fraction and only 6.5% will be present in the cheese.

AFRIKAANSE OPSOMMING: Vyf proewe is gedoen om in vitro- en in vivo-degradering, uitskeiding en absorpsie parameters van melamien (MEL) na te gaan waaroor daar min of geen inligting bekend was nie. Die eerste twee proewe was in vitro-studies, uitgevoer om die mate van MEL degradeerbaarheid in rumenvloeistof na te gaan, asook die invloed van MEL op rumen-NH3 en vlugtige vetsuur (VVS)-konsentrasies. Vir beide proewe is rumenvloeistof van lakterende, rumengekannuleerde Holsteinkoeie verkry. Vir die eerste en tweede in vitro-studies, was rumenvloeistof verkry vanaf drie en twee koeie, onderskeidelik. In albei proewe is 1 g substraat in Erlen-meyerflessies afgeweeg en 100 mL inkubasiemedium bygevoeg wat uit 20 mL rumenvloeistof en 80 mL van ‘n buffermedium bestaan het. In die eerste proef is 100 mg MEL by die substraat gevoeg, sodat die aanvanklike MEL konsentrasie in die flessies 1000 mg/L was. Die flessies is by 39° C geïnkubeer vir 0 (Kontrole), 6, 24 of 48 ure, onder streng anaerobiese kondisies. Met die beïndiging van die inkubasieperiode is 100 mL van ‘n 0.2 M perchloorsuuroplossing bygevoeg om enige melamien wat nie gedegradeer was nie, op te los. In al die proewe is melamienbepalings by wyse van LC/MSMS gedoen. Melamiendegradering was laag na 6 en 24 h inkubasie (3.2 en 5.5%, respektiewelik) en teen 48 h inkubasie het dit toegeneem tot 13.6%. In die tweede proef het die flessies 0 (Kontrole), 0.2 (T1) of 0.4 mg (T2) melamien bevat. Behandeling het geen invloed op individuele of totale VVS-konsentrasies by enige van die inkubasietye gehad nie en ook nie op NH3-konsentrasies by 6 en 48 h nie. Om een of ander onverklaarbare rede het die T2-behandeling gelei tot hoër NH3-konsentrasies by 24 h. Die gevolgtrekking is gemaak dat die byvoeging van MEL geen effek op rumen NH3-konsentrasies het nie en dat die mikroorganismes in die rumen nie daartoe in staat sal wees om MEL as ‘n stikstof-bron sal kan benut nie. In die derde proef is die uitskeiding van MEL in melkkoeie ondersoek. Vyf lakterende Holsteinkoeie is ewekansig aan vyf behandelings toegeken in ‘n 5 x 5 Latynsevierkantontwerp. Gedurende elke periode het koeie die behandelings vir 7 d ontvang, gevolg deur ‘n 8 d MEL-onttrekkingsperiode. Die eksperimentele diëte is geformuleer om ‘n daaglikse MEL-inname van 0 (M0), 500 (M1), 1000 (M2), 5000 (M3) of 10000 mg (M4) per koei/dag te verseker, toegedien via 15 kg/d van ‘n suiwelkonsentraat in pilvorm. Berekeninge gebasseer op die werk van Newton & Utley (1978) stel voor dat ‘n MEL inname van 0.16 g/kg lewende massa, geen negatiewe effek op herkouers se gesondheid sal hê nie. Dus, ‘n koei wat 600 kg weeg, sal geen skade lei deur die inname van 100 g MEL nie. In hierdie proef was die hoogste MEL behandeling (M4 = 10g/d) tien keer laer as die voorgestelde veiligheidsvlak van Newton & Utley (1978). Behandeling het geen invloed op DMI, melkopbrengs of melksamestelling gehad nie. Melamien is so gou as 8 h na eerste inname in die melk waargeneem, waarna die konsentrasie vinnig toegeneem het en ‘n piek na 3 d bereik het. Behandeling het geen invloed op die uitskeidingsdoeltreffendheid van melamien in melk gehad nie en waardes het gewissel van 1.5 tot 2.1%. Die gemiddelde skynbare verteerbaarheid van MEL was 78%. Die gemiddelde mis- en uriene-MEL-konsentrasies was 22 en 54%, onderskeidelik. Hoër melk-, mis- en uriene-MEL-konsentrasies is waargeneem namate die MEL-inhoud van die diëte gestyg het. Die gevolgtrekking is gemaak dat MEL spoedig na eerste inname in die melk verskyn en dat ‘n onttrekkingsperiode van 8 d benodig word voordat melk-, mis- en uriene-MEL onwaarneembare vlakke bereik. Uriene en mis is die primêre uitskeidingsroetes van ingenome MEL. Die vierde proef is onderneem om MEL-absorpsie in die melkklier met behulp van arterio-veneuse (A-V) verskille te ondersoek. Vyf koeie het elk 10 g MEL/d vir drie agtereen-volgende dae ontvang. Dag 3 van die proef is gekies vir bloedkolleksies aangesien vorige studies (Cruywagen et al., 2009; Shen et al., 2010; Sun et al., 2011) gewys het dat melk MEL op dag 3 van MEL inname, piek konsentrasies beryk. Vroeg gedurende die oggend van d 3 is kateters in die kaudale oppervlakkige epigastriese aar (melkaar) en die kaudale aurikulêre slagaar geplaas. Die bloedtrekkingsperiode het ‘n aanvang geneem direk nadat die koeie volledig uitgemelk is na toediening van oksitosien om te verseker dat soveel as moontlik residuele melk verwyder word. Monsters van veneuse-, sowel as arteriële bloed, is 9-uurliks geneem. Na die finale bloedtrekking is oksitosien weer toegedien, die kateters is versigtig verwyder en die koeie is direk daarna weer gemelk. Al die bloedmonsters is gesentrifugeer en plasmamonsters is ontleed vir MEL, asook vir aminosuursamestelling ten einde bloedtoevoer na die uier te bereken. Die positiewe fluks (bereken van A-V verskil) het bevestig dat netto MEL absorpsie in die melkklier plaasvind, met ‘n doeltreffendheid van 0.29%. Melamienuitskeiding in die melk was teen ‘n tempo van 5.63 mg/h. Die gemiddelde plasma- en melk-MEL konsentrasies was 5.2 en 3.9 mg/kg, onderskeidelik. Die uitskeidingsdoeltreffendheid van MEL na melk, uitgedruk as persentasie van ingenome MEL, was 1.47%. Die gevolgtrekking is gemaak dat MEL wat deur koeie ingeneem word, tot netto MEL-absorpsie in die melkklier sal lei, maar dat die absorpsiedoeltreffendheid baie laag is. In die finale proef is daar gepoog om die invloed van fermentasieprosesse gedurende die vervaardiging van kaas, joghurt en kefir op die produkte se melamieninhoud na te gaan indien die produkte van melamienbevattende melk gemaak sou word. ‘n Tweede doel van hierdie proef was om te bepaal of MEL in kaas gedegradeer kan word tydens rypwording. Kaas, joghurt en kefir is gemaak van melk wat ‘n MEL-inhoud van 6.77 mg/kg gehad het. Die kaas is vervolgens vir twee weke by 6° C rypgemaak. Die MEL-inhoud van die joghurt en kefir was 6.76 en 6.78 mg/kg, onderskeidelik, wat daarop dui dat die onderskeie fermentasieprosesse wat tydens die bereiding van joghurt en kefir plaasvind, geen invloed op hul MEL-inhoud gehad het nie en dat MEL nie gedurende hierdie kort fermentasieperiodes gedegradeer is nie. Die persentasie MEL na wei en kaas versprei was 97.4 en 6.5%, onderskeidelik. Die gevolgtrekking is gemaak dat die verskillende fermentasieprosesse betrokke tydens die vervaardiging van joghurt en kefir wat van melamienbesmette melk gemaak word, nie die MEL-konsentrasie verlaag het nie. Tydens die vervaardiging van kaas, word die MEL hoofsaaklik na die weikomponent versprei en baie min na kaas. Melamien word ook nie in kaas afgebreek gedurende ‘n verouderingsproses van twee weke nie. Die finale gevolgtrekkings is gemaak dat MEL maklik deur melkkoeie geabsorbeer word en dat die hoof uitskeidingsroete via urine is. Die uier het ‘n lae affiniteit vir MEL absorpsie en ongeveer 2% van ingenome MEL is in die melk uitgeskei. Wanneer kaas van MEL besmette melk gemaak word, sal die meerderheid van die MEL in die weifraksie konsentreer, met slegs 6.5% teenwoordig in die kaas.

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